Cell rolling separation

ABSTRACT

The present invention provides systems for cell separation based on cell rolling on surfaces along edges of regions coated with cell adhesion molecules. A variety of designs of coated regions and edges are disclosed.

RELATED APPLICATION INFORMATION

The present application is a divisional of U.S. patent application Ser.No. 14/667,615, filed Mar. 24, 2015, now U.S. Pat. No. 9,555,413, whichis a divisional of U.S. patent application Ser. No. 12/680,249, filedJun. 22, 2010, now U.S. Pat. No. 8,986,988, filed Mar. 24, 2015, whichis a U.S. National 371 Application of International Application No.PCT/US2008/078204, filed Sep. 29, 2008, which claims priority toProvisional Application No. 60/975,813, filed Sep. 27, 2007. Each ofthese applications is herein incorporated by reference in its entirety.

GOVERNMENT RIGHTS

This invention was made with U.S. government support under Grant Nos.RO1 DE016516 awarded by National Institutes of Health. The U.S.government has certain rights in the invention.

BACKGROUND

Cell rolling is an important physiological and pathological process thatis used to recruit specific cells in the bloodstream to a target tissue.For example, cell rolling along vascular endothelium in viscous shearflow is of primary biological importance, given its role in recruitmentof leukocytes to sites of inflammation, homing of hematopoieticprogenitor cells after intravenous injection, tumor cell metastasis andother inflammatory processes.

Cell rolling is a receptor-ligand mediated event that initiates anadhesion process to a target tissue through a reduction in cellvelocity. Cell rolling is typically followed by activation, firmadhesion, and transmigration. The rolling response is primarily mediatedby a family of transmembrane glycoprotein receptors called selectins,which are expressed on the surfaces of leukocytes and activatedendothelial cells. Selectins bind to carbohydrates via a lectin-likeextracellular domain. The broad family of selectins is divided intoL-selectin (CD62L), E-selectin (CD62E), and P-selectin (CD62P).L-selectin (74-100 kDa) is found on most leukocytes and can be rapidlyshed from the cell surface. E-selectin (100 kDa) is transientlyexpressed on vascular endothelial cells in response to IL-1 beta andTNF-alpha. P-selectin (140 kDa) is typically stored in secretorygranules of platelets and endothelial cells.

For example, the adhesion mechanism that mediates leukocyte rolling onthe vascular endothelium is often referred to as cell rolling. Thismechanism involves the weak affinity between P-selectin and E-selectin(expressed on vascular endothelial cells) and selectin-bindingcarbohydrate ligands (expressed on circulating hematopoietic stem cells(HSC) and leukocytes). Once ‘captured’, cells roll slowly over thesurface, in contrast to uncaptured cells, which flow rapidly in the bulkfluid.

SUMMARY

The present invention encompasses the finding that the direction ofmotion of rolling cells can be altered by altering the arrangement ofmolecules on surfaces on which cells roll. In particular, we havedemonstrated that cells may be diverted from the direction of flow usingan edge between a region coated with molecules that facilitate cellrolling and an uncoated area (e.g., see FIG. 1C). In certainembodiments, a stagnation line of no flow may act in lieu of or inaddition to such an edge to facilitate cell rolling at an angle to thedirection of flow.

The inventions described herein take advantage of these findings toprovide systems for cell rolling-based separation. Separated cells maybe used for any purpose, including without limitation diagnostic ortherapeutic purposes.

In some aspects, methods are provided that may be useful for cellseparation applications.

In certain embodiments, methods comprise providing a surface that is atleast partially coated with an ordered layer of cell adhesion molecules,wherein the surface comprises at least one edge between an area coatedwith the ordered layer and another area that is not coated with theordered layer; and flowing a population of cells across the surface in adirection which forms a non-zero angle α_(s) with the at least one edge.In such methods, at least one cell in the population of cells comprisesa surface moiety that is recognized by the cell adhesion molecules andat least one cell in the population of cells rolls for a period of timein a direction that is a, to the direction of flow as a result ofinteracting with at least a portion of the at least one edge.

In certain embodiments, methods comprise providing a three dimensionalsurface that is at least partially coated with an ordered layer of celladhesion molecules, and flowing a population of cells across the surfacein such conditions to create a stagnation line of no flow. In suchembodiments, the direction of flow forms a non-zero angle α_(s) with thestagnation line, at least one cell in the population of cells comprisesa surface moiety that is recognized by the cell adhesion molecules, andat least one cell in the population of cells rolls at least part of thetime in a direction that is α_(s) to the direction of flow.

In some aspects, provided are devices for cell separation comprising aseparation flow chamber, an inlet for flowing cells into the separationflow chamber, and an outlet for flowing cells out of the separation flowchamber. In such devices, the separation flow chamber comprises asurface that is at least partially coated with an ordered layer of celladhesion molecules, wherein the surface comprises at least one edgebetween an area coated with the ordered layer and another area that isnot coated with the ordered layer. In such devices, when cells areflowed through the inlet to the outlet, they flow at an angle α_(s) tothe direction of the at least one edge.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows P-Selectin immobilized on a polystyrene substrate usingmicrofluidic technology to create edges followed by adsorption ofBSA-FITC to reveal the design. Stripes were 100 μm wide. FIG. 1B shows Ttracks of rolling HL-60 cells which were flowed at concentration of1×10⁶ cells/mL over the substrate at a shear rate of 2 dyn/cm². Trackswere obtained by processing 194 images acquired at 0.5 Hz using a Matlabcode. Cells can be seen to interact and roll only on the selectinstripe. FIG. 1C shows a magnified image of the inset showingrepresentative tracks reveals that cells roll in the direction of fluidflow within the P-selectin stripe, but change direction and roll alongthe edge upon encountering the edge (marker ◯). Cells within theP-Selectin stripe that do not encounter the edge (marker □) roll in thedirection of the fluid flow, and not in the direction of the stripe.Other cells can be seen rolling on the edge (marker x). The direction ofcell rolling is determined by the edge, and not by the shape of thecoated area on which the cells roll.

FIG. 2 depicts a schematic of an example of an edge design that wouldresult in net displacements of two cell types in opposite directions.The surfaces comprises two different kinds of edges that make differentangles with respect to the direction of flow. The first edge encounteredby the cells makes an angle such that both cell types can follow it. Thesecond edge is inclined at a larger angle or has receptors such thatonly one cell type (dashed line) can roll along that edge. A spatialvariation in the above repeating design obtained by changing the secondedge gradually over a large area can be used for focusing of aparticular cell type.

FIG. 3A illustrates that cell separation may be performed using flowchambers with selectin edges at varying angles FIG. 3B illustrates thatseparation be performed with edges at a constant angle. Chamber length(L), width (w), cell inlet width (w_(inlet)), and chamber height (h) aredesign parameters that may be particularly relevant. As shown, in oneembodiment, ten devices may be used in parallel for cell separation toincrease throughput as shown in FIG. 3C.

FIG. 4A-D depicts different design schemes that make use of the edgeeffect. FIG. 4A shows negative selection of rolling cells away fromcells that do not follow an edge. FIG. 4B shows edges to arrange cellsin single files. FIG. 4C shows isolation of single cells byincorporation of microwells (which can also be adhesive patches tocapture cells). FIG. 4D shows adhesive areas leading to edges forenabling cells to roll before encountering the edge.

FIG. 5 is a schematic showing design parameters for selectin/mAbarrangements that comprise receptor bands of width (w) with edges makingan angle (α_(s)) with respect to the direction of flow. Also depicted isa possible path of a rolling cell that encounters the edge, follows itfor a distance, and subsequently detaches from it.

FIGS. 6A and 6B depict three dimensional surfaces which comprise edgeson which cells can be made to roll. FIG. 6A shows a curved surface. FIG.6B shows a surface with periodic bumps. Such surfaces may or may notcreate stagnation lines; nevertheless, they may influence the directionof cell rolling through the edges.

FIG. 7A shows a microchannel (of a PDMS device bonded on a glass slide)filled with water may be difficult to see by eye due to lack ofscattered light (top slide). A similar microchannel filled with cells iseasily visualized and distinguished by light scattered by the cells(bottom slide). FIG. 7B shows a magnified view of the inset (marked by asquare in FIG. 7A) showing cells trapped in the microchannel.

FIG. 8 depicts a reaction scheme of covalent immobilization ofP-selectin on an epoxy functionalized glass substrate. P-selectin isimmobilized on top of a layer of polyethylene glycol pre-immobilized onthe surface.

FIG. 9A shows still images and date on cell rolling on covalentlyimmobilized vs. physisorbed P-selectin. A comparison of number of cellsrolling on the surfaces after 28 days physisorbed versus FIG. 9Bcovalently immobilized P-selectin. In FIG. 9C, rolling dynamics ofneutrophils on P-selectin-coated surfaces under shear flow are shown.2.5×10⁵/mL of neutrophil solution was perfused on a 3 or 28-day-oldP-selectin-surface under wall shear stresses from 1 to 10 dyn/cm². Cellswere counted as rolling cells if their velocity was below 50% of freeflow velocity. Note that a significantly larger number of cells roll onthe surface with covalently immobilized P-selectin as compared to thesurface with physisorbed P-selectin.

FIG. 10A depicts schematic diagrams of P-selectin immobilization onmixed self-assembled monolayers (SAMs) of OEG-COOH/OEG-OH at differentratios using the EDC/NHS chemistry and FIG. 10B shows mixed SAMs ofOEG-biotin/OEG-OH after biotinylation of P-selectin through conjugationthrough —SH group of P-selectin. Note that P-selectin immobilizedthrough amide bonds FIG. 10A and through biotin-streptavidin bonds FIG.10B should have random and oriented conformation on the surfaces,respectively.

FIG. 11A shows Surface plasmon resonance (SPR) sensorgrams of P-selectinimmobilization with density controlled. By changing the ratio betweenOEG-COOH and OEG-OH, the amount of P-selectin immobilized is controlledand is proportional to the concentration of OEG-COOH. FIG. 11B showseffect of P-selectin orientation on antibody binding by comparison ofantibody binding on the unoriented P-selectin (EDC/NHS chemistry) andoriented P-selectin (thiol specific biotin-streptavidin chemistry). Notethat the amounts of immobilized P-selectin were comparable for eachother (˜12 nm wavelength shift (˜180 ng/cm²) for both surfaces).

FIGS. 12A-12E depicts a schematic and an image illustratingimmobilization of P-selectin to create edges. A silicone rubber mask wasplaced on a glass substrate as shown in FIG. 12A, and P-selectin wascoated on the exposed area of the substrate by physisorption as shown inFIG. 12B. The silicone mask was then removed from the substrate shown inFIG. 12C, and BSA was used to block the areas that were not coated withP-selectin as shown in FIG. 12D. Use of fluorescein-labeled BSA enabledvisualization of the P-selectin arrangement using an epifluorescencemicroscope as shown in FIG. 12E. HL-60 cells adhered selectively to theP-selectin region, confirming coating of some areas of the substratewith P-selectin. Scale bar: 100 μm

FIG. 13A-13D shows photographs illustrating that a P-selectin edgedirects motion of rolling cells. FIG. 13A is a photograph at t=0seconds. FIG. 13B was taken at t=50 seconds. FIG. 3C was taken at t=100seconds and FIG. 13D was taken at t=150 seconds. Rolling HL-60 cellsthat encountered the edge of a P-selectin-coated area making an angle tothe fluid flow direction were forced to roll along the edge. The motionof a cell forced to roll along the edge is compared with another cellrolling in the direction of fluid flow, highlighted by circles. The edgesucceeded in changing the direction of motion of the rolling cell by8.6°, resulting in effectively displacing the cell by 0.15 mm from itsoriginal position for every 1 mm of length along the direction of flow.Wall shear stress was 1.9 dyn/cm².

FIG. 14A shows results from analyses of cell and microsphere rolling.Matlab tracking of rolling cells generated from a set of 236 imagesclearly shows the effect of the edge. Inability of cells to cross overthe edge resulted in higher density of tracks at the edge. Cell rollingwas observed in the P-selectin coated region (pink) but not in theblocked region (white). Scale bar: 300 μm. FIG. 14B shows longer (>300μm) tracks of cells rolling on the edge and inside the P-selectin regionclearly show that the edge affected the rolling direction. Scale bar:300 μm. FIG. 14C shows angular distribution histogram of the directionof travel of cells rolling near the edge (red) with respect to thoseaway from the edge (blue). Wall shear stress was 1.9 dyn/cm2 (0.19 Pa,300 μL/min). (D) Similar experiments done with 9.96 urn diameter sLexcoated microspheres that roll on P-selectin reveal that the edge did nothave a large effect on microspheres as their direction of travel did notchange substantially. Wall shear stress was 0.33 dyn/cm² (0.03 Pa, 200μL/min).

FIG. 15A illustrates a potential mechanism of cell rolling along aselectin edge. Bonds on the trailing edge experience maximum strain.When these bonds break, it results in an asymmetric rotation of the cellas shown in FIG. 15B that causes the cell to move along the edge asshown in FIG. 15C. This mechanism is similar to cell rolling on asurface, but in addition to rotation along an axis parallel to thesurface, the cell may also spin in plane along an axis perpendicular tothe surface as shown in FIG. 15B. In the case of a rigid microsphere,the area of contact is small and the force due to the flow acts througha point vertically above the area of contact and this asymmetric motionbecomes difficult.

FIG. 16A depicts a microfluidic device for separation of cells byrolling along receptor edges with a flow channel height of 30 urn. FIG.16B depicts a stream of assorted fluorescent microspheres (2-30 μmdiameter) and HL-60 cells and a buffer stream was injected into thedevice that contained arrangements of P-selectin at an angle to thefluid stream. Rolling HL-60 cells were selectively diverted andseparated away from the microspheres in original stream, evident in thecomposite fluorescence and bright field image. Dashed line outlines theboundary between the cell and microsphere stream and the buffer stream.The arrow indicates direction of cell rolling along the edge. Cells onthe edge are circled. Scale bar: 100 μm.

FIG. 17A depicts a design of micro-device. In FIG. 17B HL-60 cellrolling tracks show control of rolling using edges of P-selectin coatedareas (pink). FIG. 17C shows cells within collection channels observedwith low power microscope.

FIG. 18 illustrates how microfluidic arrangements of biomolecules can beachieved by flowing the biomolecules through PDMS microchannelsreversibly bonded to a substrate. This technique has been used to createP-selectin edges and the design has been visualized by exposure tofluorescently labeled BSA following the arranging step. BSA selectivelyadsorbs on the region without P-selectin and appears bright.

FIG. 19 depicts a schematic for site density determination of CD64 onneutrophils. To determine the site density of antibodies bound to thesurface of the neutrophils, standard IgG beads will be bound to theFITC-biotin antibody and used to generate a calibration curve of sitedensity versus fluorescence intensity as described by the supplier.

FIG. 20A shows a schemative for a device that could be used to sortactivated CD64⁺ neutrophils from non-activated (CD64⁻) neutrophils.Activated neutrophils are expected to be distinguishable fromnon-activated neutrophils FIG. 20B as they travel at a different angleand exit through outlet FIG. 20A. Non-activated neutrophils exit throughoutlet FIG. 20B. Activated neutrophils may be detected from a shift inthe relative distribution of cells at the two outlets.

FIG. 21A shows examples of surfaces that can be used to createstagnation lines for three-dimensional cell separation applications. Acylinder and FIG. 21B a ridge can be coated on their outer surfaces withcell adhesion molecules and used in three-dimensional cell rolling-basedseparation systems. Arrows indicate streamlines of fluid flow.Stagnation lines represent regions of no flow in the near vicinity ofthe surface.

DEFINITIONS

Throughout the specification, several terms are employed that aredefined in the following paragraphs.

The terms “about” and “approximately,” as used herein in reference to anumber, generally includes numbers that fall within a range of 5%, 10%,or 20% in either direction of the number (greater than or less than thenumber) unless otherwise stated or otherwise evident from the context(except where such number would exceed 100% of a possible value).

The phrase “adhesive patch” as used herein refers to a region (such as,for example, on a surface) onto which molecules to which cells canadhere are arranged. Such adhesive molecules generally can comprise anyligands with stronger interactions with cells than cell adhesionmolecules. Examples of such molecules include antibodies and antibodyfragments. The density of such molecules in the adhesive patch (or thedimensions of the patch) may in some embodiments be controlled such thatcells encountering the patch slow down but do not stop. In someembodiments, the density of molecules in the adhesive patch (or thedimensions of the patch) is controlled such that cells encountering thepatch stop.

The term “adsorb” is used herein consistently with its generallyaccepted meaning in the art, that is, to mean “to collect byadsorption.” “Adsorption” refers to the process by which specificgasses, liquids or substances in solution adhere to exposed surfaces ofmaterials, usually solids, with which they are in contact.

The term “cell adhesion molecule,” as used herein, generally refers toproteins located on cell surfaces involved in binding (via celladhesion) of the cell on which it is found with other cells or with theextracellular matrix. Examples of cell adhesion molecules include, butare not limited to, full-length, fragments of, analogs of, and/ormodifications of selectins (e.g., E-selectins, P-selectins, L-selectins,etc.), integrms (e.g., ITGA4, etc.), cadherins (e.g., E-cadherins,N-cadherins, P-cadherins, etc.), immunoglobulin cell adhesion molecules,neural cell adhesion molecules, intracellular adhesion molecules,vascular cell adhesion molecules, platelet-endothelial cell adhesionmolecules, L1 cell adhesion molecules, and extracellular matrix celladhesion molecules (e.g., vitronectins, fibronectins, laminins, etc.).As used herein, the term “cell adhesion molecule” also encompasses othercompounds that can facilitate cell adhesion due to their adhesiveproperties. In some embodiments of the invention, aptamers,carbohydrates, peptides (e.g., RGD (arginine-glycine-aspartate)peptides, etc.), and/or folic acid, etc. can serve as cell adhesionmolecules. As used herein, such compounds are encompassed by the term“cell adhesion molecule.” As used herein, terms referring to celladhesion molecules including, but not limited to, “cell adhesionmolecule,” “selectin,” “integrin,” “cadherin,” “immunoglobulin celladhesion molecule,” “neural cell adhesion molecules,” “intracellularadhesion molecules,” “vascular cell adhesion molecules,”“platelet-endothelial cell adhesion molecules,” “L1 cell adhesionmolecules,” “extracellular matrix cell adhesion molecules,” encompassfull length versions of such proteins as well as functional fragments,analogs, and modifications thereof, unless otherwise stated. Likewise,terms referring to specific cell adhesion molecules including, but notlimited to, “E-selectin,” “P-selectin,” “L-selectin,” “JTGA4,”“E-cadherin,” “N-cadherin,” “P-cadherin,” “victronectin,” “fibronectin,”“laminin,” etc., also encompass full length versions of such proteins aswell as functional fragments, analogs, and modifications thereof, unlessotherwise stated. As used herein, the term “cell adhesion molecule” doesnot encompass antibodies.

The phrase “cell culture,” is used herein to refer to the growing ofcells, typically in a controlled environment. Such cells can be derivedfrom multicellular eukaryotes, especially animal cells, or can bemicroorganisms such as bacteria. The term “tissue culture” is often usedinterchangeably with the term “cell culture” when the cells are derivedfrom multicellular eukaryotic animals.

The term “cell modifying ligand,” as used herein, generally refers tomolecules that are capable of modifying the biological behavior of acell. For example, a protein that triggers a molecular signal within acell (e.g., expression of another protein) is a cell modifying ligand.

The term “deformability,” as used herein, where it refers to cells,means the ability of cells to change their shape, such as, for example,as they pass through narrow spaces, as they roll along a surface, etc.

The term “linker,” as used herein, refers to a chemical moiety used toattach a group or moiety (e.g., a cell adhesion molecule) to anotherfunctional group (such as, for example, a functional group immobilizedon a surface). Without limitation, in some embodiments, the linkermoiety comprises one or more of a dextran, a dendrimer, polyethyleneglycol (PEG), poly(L-lysine), poly(L-glutamic acid), poly(D-lysine),poly(D-glutamic acid), polyvinyl alcohol, and polyethylenimine. In someembodiments, the linker moiety comprises one or more of an amine, analdehyde, an epoxy group, a vinyl, a thiol, a carboxylate, and ahydroxyl group. In some embodiments, the linker moiety includes a memberof a ligand/receptor pair and the cell surface molecule has beenchemically modified to include the other member of the pair.

The phrase “mesenchymal stem/progenitor cell” (abbreviated “MSPC”), asused herein, refers to self-renewing and multipotent cells that aredistributed in a variety of adult and fetal tissues including the bonemarrow, skin, kidney, lung and liver. MSPCs can be maintained andpropagated in culture prior to directing the differentiation intomultiple cell types including adipocytes, chondrocytes, osteoblasts,hepatocytes, and cardiomyocytes. Bone marrow and adipose tissue are themost abundant sources of MSPCs. The phrase is used interchangeably with“mesenchymal stem cell” (abbreviated “MSC”).

The term “oriented,” as used herein, is used to describe molecules(e.g., cell adhesion molecules, etc.) having a definite or specifiedspatial orientation, that is, a non-random orientation. For example,cell adhesion molecules are “oriented” on a surface if a substantialportion of the cell adhesion molecules on the surface have a particularspatial orientation with respect to the surface. In certain embodimentsof the invention, the “substantial portion” comprises at least 50% ofthe molecules on the surface.

The term “unoriented,” as used herein, is used to describe molecules(e.g., cell adhesion molecules, etc.) having no particular or specifiedorientation, that is, a random orientation. For example, cell adhesionmolecules may be described as “unoriented” on a surface if the celladhesion molecules generally do not have a defined orientation withrespect to the surface.

The term “ordered layer,” as used herein, refers to a layer having aproperty which is substantially uniform, periodic, and/or patternwiseover at least 50% of the layer. In some embodiments, an ordered layerhas one or more features chosen from a substantially uniform density anda substantially uniform spatial orientation of the cell adhesionmolecules. In some embodiments, an ordered layer has one or morefeatures chosen from a patternwise distribution, a patternwise density,and a patternwise spatial orientation of the cell adhesion molecules. Insome embodiments, the ordered layer of cell adhesion molecules allows avelocity of cell rolling over the ordered layer that is substantiallyproportional to the shear stress applied to the ordered layer.

The term “physisorb” is used herein consistently with its generallyaccepted meaning in the art, that is, “to collect by physisorption.”“Physisorption” refers to adsorption that does not involve the formationof chemical bonds.

The phrase “progenitor cell” as used herein refers to cells that have acapacity to differentiate into a specific type of cell. The termgenerally refers to cells that are further differentiated along aparticular lineage than stem cells.

The term “self-assembled monolayer” (abbreviated as “SAM”), as usedherein, refers to a surface comprising a single layer of molecules on asubstrate that can be prepared by adding a solution of the desiredmolecule onto the substrate surface and washing off the excess.

The phrase “stagnation line,” as used herein, refers to a region of zeroflow velocity near a surface of an object where flows on the surfaceconverge from different directions. The shear along the stagnation lineis zero, and the flow velocity close to the surface defines a planepassing through the stagnation line. In this plane, the flow velocitymust make an angle other than 90 degrees with respect to the stagnationline. (The angle is 90 degrees in the case of vertical posts).

The phrase “stem cell” as used herein refers to cells that are capableof self renewal through mitotic cell division and are capable ofdifferentiating into a diverse range of specialized cell types. Examplesof stem cells include, but are not limited to, mesenchymal stem cells,hematopoietic stem cells, and embryonic stem cells.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

As mentioned above, the present invention provides systems useful forcell separation by employing cell rolling across a surface.

I. Methods

Provided are methods comprising steps of providing a surface at leastpartially coated with an ordered layer of cell adhesion molecules andflowing a population of cells across the surface. Surfaces comprise atleast one edge between an area coated with the ordered layer and an areathat is not coated with the ordered layer. Populations of cells areflowed across surfaces in a direction that together with at least oneedge form a non-zero angle α_(s). At least one cell in the population ofcells comprises a surface moiety that is recognized by the cell adhesionmolecules, and rolls at least part of the time in a direction α_(s) tothe direction of flow as a result of interacting with the edge. In someembodiments, such cells roll along the edge at least part of the time.

In certain embodiments, methods further comprise separating the at leastone cell from certain cells in the population. In some embodiments,cells comprising the surface moiety roll on the coated area, but at adistance which is at least one cell diameter away from the edge. In suchembodiments, the cells may roll at an angle which is smaller than α_(s).In some such embodiments, the angle smaller than α_(s) is orapproximates zero. That is, cells that do not interact with the at leastone edge roll in the direction of flow.

A. Coated Surfaces

Surfaces are generally partially coated with an ordered layer of celladhesion molecules and may or may not comprise additional molecules asdiscussed herein.

Cell Adhesion Molecules

A variety of cell adhesion molecules can be used in the practice ofcertain embodiments of the present invention. In some embodiments, thelayer of cell adhesion molecules comprises cell adhesion moleculeshaving a dissociation constant (K_(D)) for interaction with one or morecell surface moieties (e.g., proteins, glycans, etc.) that is greaterthan about 1×10⁻⁸ mole/liter (M). In some embodiments, the layer of celladhesion molecules comprises cell adhesion molecules having adissociation constant (K_(D)) for interaction with one or more cellsurface molecules that is in the range of about 1×10⁻⁴ molar to about1×10⁻⁷ M, inclusive. It will be appreciated that the behavior of cellson the coated surface will depend in part on the dissociation constant.

In general, any cell adhesion molecule may be used. Examples of celladhesion molecules useful in certain embodiments of the presentinvention include, but are not limited to, full-length, fragments of,analogs of, and/or modifications of selectins (e.g., E-selectins,P-selectins, L-selectins, etc.), integrins (e.g., ITGA4, etc.),cadherins (e.g., E-cadherins, N-cadherins, P-cadherins, etc.),immunoglobulin cell adhesion molecules, neural cell adhesion molecules,intracellular adhesion molecules, vascular cell adhesion molecules,platelet-endothelial cell adhesion molecules, L1 cell adhesionmolecules, and extracellular matrix cell adhesion molecules (e.g.,vitronectins, fibronectins, laminins, etc.). In some embodiments,aptamers, carbohydrates, peptides (e.g., an RGD peptide), folic acid,etc. can serve as cell adhesion molecules. The layer of cell adhesionmolecules may include a single cell adhesion molecule or a combinationof different kinds of cell adhesion molecules.

Cell adhesion molecules may be bound to surfaces in a variety of ways.Noncovalent interactions such as, for example, van der Walasinteractions, hydrogen bonding, and electrostatic interactions (alsoknown as ionic bonding) etc. may be used.

Covalent bonds may also be used. Any covalent chemistry may be used tocovalently attach cell adhesion molecules to a substrate surface. Thoseskilled in the art will appreciate that the methods described in theExamples are exemplary and could be readily modified based on knowledgein the art. In some embodiments, cell adhesion molecules are attached toa surface through one or more linker moieties. In some embodiments, alinker moiety is bound to the cell adhesion molecule at one of its endsand to the surface of the substrate at another end. In general, the bondbetween the linker moiety and the surface is covalent. The bond betweenthe linker moiety and the cell adhesion molecule may be covalent ornon-covalent (e.g., if it involves a ligand/receptor pair as discussedherein). Without limitation, in some embodiments, the linker moietycomprises one or more of a dextran, a dendrimer, polyethylene glycol(PEG), poly(L-lysine), poly(L-glutamic acid), poly(D-lysine),poly(D-glutamic acid), polyvinyl alcohol, and polyethylenimine. In someembodiments, the linker moiety comprises one or more of an amine, analdehyde, an epoxy group, a vinyl, a thiol, a carboxylate, and ahydroxyl group. In some embodiments, the linker moiety includes a memberof a ligand/receptor pair and the cell surface molecule has beenchemically modified to include the other member of the pair.

In addition to improving the long term stability and behavior of thecoated surface, the use of covalent bonding instead of physisorption,enables one to control the density, arrangement and orientation of celladhesion molecules on the substrate surface. For example, the densitywill depend on the density of groups on the surface which are availablefor covalent bonding. Similarly, the arrangement will depend on thearrangement of groups on the surface which are available for covalentbonding. Methods are well known in the art for preparing surfaces withdifferent densities and arrangements of suitable groups for covalentbonding (e.g., see Rusmini et al. Protein immobilization strategies forprotein biochips. Biomacromolecules 2007 June; 8(6): 1775-89. andLeckband et al. An approach for the stable immobilization of proteins.Biotechnology and Bioengineering 1991; 37(3):227-237, the entirecontents of both of which are incorporated herein by reference). In someembodiments, the density of cell adhesion molecules ranges from about 10ng/cm² to about 600 ng/cm². In some embodiments, the density of celladhesion molecules is greater than about 30 ng/cm². For example, in someembodiments, the density of cell adhesion molecules ranges from about 30ng/cm² to about 360 ng/cm². In some embodiments, the density of celladhesion molecules ranges from about 50 ng/cm² to about 300 ng/cm². Insome embodiments, the density of cell adhesion molecules ranges fromabout 100 ng/cm² to about 200 ng/cm².

In some embodiments, the orientation of cell adhesion molecules on thesurface is controlled. This can be advantageous, e.g., because the celladhesion molecules are forced to interact with cells only if aparticular region of the cell adhesion molecules is accessible to thecells. For example, P-selectin includes a single cysteine residue. As aresult, if P-selectin is attached to the surface via a linker moietythat reacts specifically with cysteine, all P-selection molecules willbe attached to the surface with the same orientation. In general, thisapproach can be applied whenever the cell adhesion molecule includes aunique group. In some embodiments, a cell adhesion molecule can beengineered or chemically modified using methods known in the art toinclude such a unique group (e.g., a particular amino acid residue) at aposition that provides an optimal orientation. For example, a suitableamino acid residue can be added at the C- or N-terminus of protein basedcell adhesion molecules.

In some embodiments, the cell adhesion molecules are synthesized and/orpurified such that only a limited subset of the residues is able toreact with reactive groups on the surface or on the linker. In someembodiments, there is only one group or residue on each cell adhesionmolecule that can react with reactive groups on the surface or on thelinker. For example, in some embodiments, cell adhesion molecules aresynthesized and/or purified with protecting groups that prevent theresidues to which they are attached from reacting with reactive groupson the surface or linker. In such embodiments, one or more residues inthe cell adhesion molecule are not protected. Because the cell adhesionmolecule can only attach to the surface or linker via the one or moreunprotected residues, the cell adhesion molecule may attach to thesurface or linker in a specific orientation. In some embodiments, theprotective groups are removed after attachment of the cell adhesionmolecule to the surface or linker. (See, e.g., Gregorius et al.Analytical Biochemistry 2001 Dec. 1; 299(1):84-91, the entire contentsof which are incorporated herein by reference.)

Antibodies

In some embodiments, antibodies (including antibody fragments) may beco-immobilized with cell adhesion molecules. In general, an antibody maybe attached to the surface in a similar fashion to the cell adhesionmolecule (e.g., using the same linker moiety). In certain embodiments,the antibody may be attached using a different covalent attachmentmethod. In certain embodiments, the antibody may be attachednon-covalently. In certain embodiments, the ordered layer comprises atleast one antibody that is covalently attached to the surface and leastone antibody that is non-covalently attached to the surface.

In certain embodiments, an antibody that binds to a cell surface moietymay be coimmobilized with cell adhesion molecules. In principle, anypair of antibody and surface ligand may be used in accordance with theinvention, so long as the antibody binds to the surface ligand. Forexample, if it is desired to modify interactions between the coatedsurface and a cell type that expresses CD64, anti-CD64 antibodies may becoimmobilized with cell adhesion molecules. Those skilled in the artwill appreciate how this can be extended to other surface ligands thatare known in the art. Molar ratios of cell adhesion molecules toantibodies in such embodiments may be varied depending on the desiredrolling characteristics (such as, for example, velocity, percentage ofcells stopping, etc.). Examples of suitable ratios include those rangingfrom about 100:1 to 1:100. In some embodiments, molar ratios rangebetween 20:1 and 1:1.

In some embodiments, antibodies can be included in order to adjust thespeed at which cells roll on a coated surface. In some embodiments thismay be achieved by controlling the density and/or arrangement ofantibodies. In some embodiments, antibodies may be immobilized ontosurfaces at such a density as to slow down the speed of rolling withoutcausing the cells to stop. In some embodiments, antibodies may bearranged onto surfaces at such a density as to cause cell rolling tostop.

Cell Modifying Ligands

In some embodiments, cell modifying ligands may be co-immobilized withcell adhesion molecules. In general, a cell modifying ligand may beattached to the surface in a similar fashion to the cell adhesionmolecule (e.g., using the same linker moiety). In certain embodiments,the cell modifying ligand may be attached using a different covalentattachment method. In certain embodiments, the cell modifying ligand maybe attached non-covalently. In certain embodiments, the ordered layercomprises at least one cell modifying ligand that is covalently attachedto the surface and least one cell modifying ligand that isnon-covalently attached to the surface.

In some embodiments, the population of cells which is flowed over acoated surface includes at least one subpopulation of cells with acommon characteristic, and the cell modifying ligand is capable ofmodifying a phenotype of the subpopulation of cells. Any of a variety ofcell types can comprise the subpopulation, as discussed herein. As anexample, certain cancer cells may express a receptor such as TNFreceptor 5 and/or 6, which is not expressed on normal cells. Tumornecrosis factor (TNF)-related receptor apoptosis-inducing ligand (TRAIL)specifically binds to TNF receptors 5 and 6. To induce apoptosis orprogrammed cell death of such cells, TRAIL may be co-immobilized with acell adhesion molecule. Cell modifying ligands such as TRAIL and/orother chemotherapeutic agents can be co-immobilized with a cell adhesionmolecule to impart signals to kill or arrest growth of cancer cells. Itwill be appreciated by those skilled in the art that other cellmodifying ligands can be immobilized and/or presented on and/or withinthe substrate to influence the behavior of cells that interact with thecell adhesion molecules. For example, fibroblast growth factor 2 (FGF-2)can be presented to facilitate maintaining cells in an undifferentiatedstate. As a further example, bone morphogenic protein 2 (BMP-2) can bepresented to stimulate osteogenic differentiation of stem cells, etc.Combinations of cell modifying ligands can also be used together.

B. Designs

In general, coated surfaces comprise at least one edge between a coatedarea and an uncoated area. The is no limitation on the types of designswhich may be used in order to achieve one or more edges.

Edge(s)

At least one edge on the surface generally forms a non-zero angle α_(s)with the direction of flow. In certain embodiments, α_(s) is at least0.5 degrees. α_(s) may be, in various embodiments, at least 1 degree, atleast 2 degrees, at least 3 degrees, at least 4 degrees, at least 5degrees, at least 6 degrees, at least 7 degrees, or at least 8 degrees.α_(s) may be, in various embodiments, less than about 70 degrees, lessthan about 65 degrees, less than about 60 degrees, less than about 55degrees, less than about 50 degrees, less than about 45 degrees, lessthan about 40 degrees, less than about 35 degrees, less than about 30degrees, less than about 25 degrees, less than about 20 degrees, or lessthan about 15 degrees.

The at least one edge may be substantially linear and/or may comprise acurved portion. In some embodiments, an edge may include both linear andcurved portions. In some embodiments of the invention, surfaces comprisea plurality of edges. In some such embodiments of the invention, atleast two of the edges form different angles to the direction of fluidflow. FIG. 2 shows one example of a design that makes use of pluralityof edges having different angles.

In certain embodiments of the invention, the edge is a sharp edge.Sharpness of an edge may be characterized by a certain percent change indensity over a given distance. When referring to edges between coatedareas and uncoated areas, it may be useful to consider densities ofmolecules (e.g., cell adhesion molecules) in the ordered layer and usethe maximum density in the coated area for comparison. “100% density”could be defined as the maximum density in the coated area adjacent tothe edge. A change in density, for example, between 10% and 90% over asmall distance indicates a sharp edge; the same change over a largerdistance indicates a blurry edge. In some embodiments of the invention,the edge is characterized by a sharpness that corresponds to a changefrom 10% to 90% density over a distance of less than about 5 μm. In someembodiments, the distance is less than about 3 μm, less than about 2 μm,less than about 1 μm, less than about 0.5 μm, less than about 0.2 μm, orless than about 0.1 μm.

Without wishing to be bound by any particular theory, it is proposedthat a certain degree of sharpness may be necessary in order to inducecell rolling along a particular direction. It is possible that at asharp edge, cells can initiate an asymmetrical motion that is onlypossible when it interacts simultaneously with a surface coated withligands that interact with the cell surface and with an uncoatedsurface.

Arrangement of Edge(s)

Areas coated with ordered layers may form any of a variety of designsthat provide edges as discussed herein. Designs may comprise a pluralityof coated areas. Some examples of designs are depicted in FIGS. 2-4.

In certain embodiments of the invention, designs comprise one or morecoated areas that each define strips having at least two edges. The twoedges of a strip may be substantially parallel; alternatively oradditionally, the strips themselves may be substantially parallel toeach other. In some embodiments wherein the strips are substantiallyparallel to each other, strips may be separated by a substantially fixeddistance w_(g) between adjacent strips and may have substantially thesame width w_(s). Both parameters w_(g) and w_(s) may be varied asappropriate, for example, to achieve cell-rolling based separation for aparticular set of conditions. For example, w_(s) may be in the range offrom about 0.01 μm to about 10 mm. In some embodiments of the invention,w_(s) is less than about 100 μm, less than about 75 μm, or less thanabout 50 μm. In some embodiments of the invention, w_(s) is greater thanabout 0.1 μm or greater than about 1 μm.

It may be useful in some circumstances to define w_(s) in relation tothe average diameter d of a cell that may be induced to roll. In someembodiments, w_(s) is less than 3d, less than 2d, or less than d.

w_(g) may be, for example, in a range from about 0.2 μm to about 10 μm.In some embodiments, w_(g) is less than about 100 μm, less than about 75μm, or less than about 50 μm. In some embodiments, w_(g) is greater thanabout 1 μm, greater than about 5 μm, or greater than about 10 μm.

w_(g) may approximately equal, be greater than, or be less than w_(s).

In certain embodiments, w_(g), w_(s), or both, may have a certainrelationship with other parameters. For example, cells may roll along anedge with a contact radius r_(contact). In some embodiments,w_(g)>r_(contact). In some embodiments, w_(g) is slightly bigger thanr_(contact), for example, w_(g) may be bigger than r_(contact) butlimited such that w_(g)<1.5 r_(contact), w_(g)<1.2 r_(contact), orw_(g)<1.1 r_(contact).

Designs may comprise strips of coated areas that are not parallel to oneanother. In some embodiments, such strips originate from a common point,or from a common area such as an inlet, and radiate outward at differentangles. One example of such a design is depicted in FIG. 3A.

Alternatively or additionally, designs may comprised coated areasdefined by shapes such as, for example, squares, rectangles, triangles,polygons, ellipses, circles, arcs, waves, and/or combinations thereof.It will be appreciated that a plurality of such shapes and/or strips maybe arranged into any design as long as the overall design provides atleast one edge with a non-zero angle to the direction of flow across thesurface. See, for example, FIGS. 2 and 4. In general, the nature of thedesign may be tailored depending on the type of cell(s) which is beingseparated and/or the type of separation which is desired. For example,when a system is needed to separate a single cell type then a simpledesign with a single type of edge may suffice. However, when a system isneeded to separate a plurality of different cell types then a morecomplex design with different types of edges may be required.

Surfaces may incorporate additional elements or features for aparticular purpose, e.g., capturing cells within the surface, asdepicted in FIG. 4C. Elements may be physical structures, such as, forexample, wells (i.e., depressions in the surface), that restrict cellsfrom flowing in the direction of flow. Similarly, adhesive patches maybe incorporated into surfaces to facilitate immobilization of cells inparticular regions on the surface. Adhesive patches may comprise, forexample, molecules such as antibodies that facilitate cells reducingtheir velocity and/or stopping. In certain embodiments, surfaces furthercomprise adhesive patches located adjacent to and/or leading to at leastone edge. In some embodiments, adhesive patches are located upstream ofa coated area. By “upstream” it is meant that the adhesive patches arelocated such that cells flowing over the surface encounter the adhesivepatches before they encounter the coated area. Such adhesive patches mayattract cells and facilitate cells rolling along the edges.

C. Cells

Populations of cells may comprise any of a variety of cell types and maybe obtained from any of a variety of sources. Cell populations typicallycomprise at least one subpopulation of cells with a commoncharacteristic.

In some embodiments of the invention, in the step of flowing, at leastone cell in the subpopulation rolls at least part of the time. Such acell may roll in a direction that is α_(s) to the direction of flow as aresult of interaction with the edge, wherein α_(s) is the angle thatforms between the edge and the direction of fluid flow. In someembodiments, substantially all cells in the subpopulation roll at leastpart of the time.

The common characteristic can be a phenotype such as expression of acell surface moiety, cell type (such as, for example, lineage type),differentiation potential, etc. For example, cells in the subpopulationmay all comprise a cell surface moiety that is recognized by the celladhesion molecules. Examples of cell surface moieties include ligands ofP-selectin, ligands of E-selectin, ligands of L-selectin, etc. Examplesof such moieties include P-selectin ligand 1 (PSGL-1), CD44 (a ligandfor E-selectin and L-selectin), glycosylation-dependent cell adhesionmolecule 1 (GlyCAM-1, a ligand for L-selectin), CD 15 (a ligand forP-selectin), CD34 (a ligand for L-selectin), E-selectin ligand 1(ESL-1), etc. Further examples of surface moieties include Very LateAntigen 4 (VLA-4, a ligand for VCAM-1), gp200, etc.

Subpopulations may comprise particular cell types and/or combinations ofcell types. For example, cells in a subpopulation may be cancer cells.Further examples of cell types include stem cells (e.g., mesenchymalstem cells, hematopoietic stem cells, embryonic stem cells, etc.),progenitor cells, red blood cells, neutrophils, lymphocytes, monocytes,white blood cells, etc. In some embodiments, all cells in asubpopulation are of a particular cell type, e.g., all cancer cells, allstem cells, all progenitor cells, etc. Though platelets are not formallyclassified cells, they may be induced to roll and separated usingsystems of the present invention.

Cells may be obtained from a variety of sources, including, but notlimited to, bodily fluids containing cells (such as, for example, blood,lymph, ascites fluid, urine, saliva, synovial fluid, cerebrospinalfluid, vitreous humor, seminal fluid, etc), tissue samples, frozenstocks, cell cultures, etc.

Cells may be treated with agents before and/or as they are flowed. Forexample, cells may be treated with agents that modify theirdeformability. Examples of such agents include cytochalasin,N-ethylmaleimide, p-choloromercuribenzene, vinblastine, etc. In certainembodiments, this treatment step may facilitate cell rolling of acertain type of cell.

D. Cell Rolling

Cells flowing close to the surface may, under appropriate conditions,roll across the surfaces of coated areas. Cells that are further awayfrom edges (for example, more than one cell diameter away from the edge)generally will continue to roll in or approximately in the direction offluid flow. In certain embodiments of the invention, cells at or nearthe edge (for example, within one cell diameter of the edge) roll alongthe edge at least part of the time. In some embodiments, cells that areaway from the edge may roll in or approximately in the direction offluid flow until they disengage from the surface or encounter an edge,at which point they may begin to roll along the edge. In someembodiments, cells rolling along an edge follow the edge for some time,disengage from the surface, reattach (for example, on another or on thesame coated area), and begin rolling again. (See, for example, FIG. 5).

It will be appreciated that under a given set of conditions, not allcells in a population of cells may roll along the edge. Cell rolling maybe selective in that only certain subpopulations of cells will roll. Asan example, populations may comprise cells that do not comprise a cellsurface moiety that is recognized by the cell adhesion molecules. Suchcells would not roll along the surface under most conditions. Amongcells in the population that do express a cell surface moiety recognizedby the cell adhesion molecules, differences may exist that arepermissible to rolling along the edge for one or more subpopulations,while not being permissible to rolling for other subpopulations. Withoutwishing to be bound by any particular theory, any of a number ofcharacteristics may serve to differentiate the subpopulations that rollalong an edge from those that do not under a given set of conditions.Such characteristics might include, for example, density of cell surfacemoieties, cell size, cell deformability, etc.

Direction

As mentioned above, cells may roll along edges, at least one of whichforms a nonzero angle α_(s) with the direction of flow. Cells may, insome embodiments, therefore roll in a direction that is α_(s) from thedirection of flow. As discussed above, a, may vary. In certainembodiments, α_(s) is at least 0.5 degrees. α_(s) may be, in variousembodiments, at least 1 degree, at least 2 degrees, at least 3 degrees,at least 4 degrees, at least 5 degrees, at least 6 degrees, at least 7degrees, or at least 8 degrees. α_(s) may be, in various embodiments,less than about 70 degrees, less than about 65 degrees, less than about60 degrees, less than about 55 degrees, less than about 50 degrees, lessthan about 45 degrees, less than about 40 degrees, less than about 35degrees, less than about 30 degrees, less than about 25 degrees, lessthan about 20 degrees, or less than about 15 degrees.

Without wishing to be bound by any particular theory, cells may rollmore easily at smaller angles and may tolerate angles up to a maximumangle α_(tr). α_(tr) may vary depending on characteristics of the cells,particular conditions of the cell separation system, etc.

Speed

Speed of cell rolling may also depend on characteristics of cells, onparticular conditions of the system, etc. The speed of a cell rollingalong an edge may, in some embodiments, be greater than the speed of asimilar cell rolling on a coated area away from the edge. A given cellmay roll with variable speed or with a substantially constant speedduring the time it rolls along an edge. In some embodiments, cellswithin a subpopulation have uniform average speeds when rolling alongedges of a given angle α_(s). In some embodiments, cells within asubpopulation have different average speeds when rolling along edges ofa given angle α_(s).

Cells may roll along an edge, for example, in a direction that is α_(s)to the direction of flow at an average speed of at least about 0.1 μm/s,at least about 0.5 μm/s, at least about 0.8 μm/s, or at least about 1.0μm/s.

Shear

Assuming a linear fluid velocity profile, shear on a cell may be relatedto fluid velocity in some embodiments as:

$\begin{matrix}{\tau = {\mu\frac{V_{fluid}}{R_{cell}}}} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$wherein μ is the viscosity of the fluid, R_(cell) is the radius of thecell, and V_(fluid) is the velocity of fluid flow at distance R_(cell)from the surface.

In some embodiments, the shear stress on cells flowed over the surfaceis in a range between about 0.05 dyn/cm² to about 50 dyn/cm². In someembodiments, the shear stress ranges between about 0.2 dyn/cm² to about5 dyn/cm².

Cell Deformability

Without wishing to be bound by any particular theory, deformability of agiven cell may influence its ability to roll along an edge. For example,in some embodiments, cells that are less deformable may be less amenableto rolling along an edge as are cells that are more deformable.

The area with which a cell contacts a surface as it rolls may give anindication of the deformability of the cell. For example, cells thatinteract with a surface with a large contact area may be more deformablethan those that do so with a small contact area. Contact area may bedefined, in some embodiments, by a contact radius r_(contact).

In some embodiments, cells rolling along an edge contact the surfacewith a cell contact radius r_(contact) of at least about 0.25 μm. Invarious embodiments, r_(contact) may be at least about 1 μm, at leastabout 2 μm, at least about 3 μm, or at least about 4 μm.

Deformability of cells may be altered, for example, by treatment beforeand/or during flowing with an agent that modifies cell deformability, asdiscussed herein.

Relationships and Combinations of Parameters

In certain embodiments of the invention, parameters are defined inrelation to each other.

In some embodiments, physical constraints guide relationships betweentwo or more parameters. For example, a, may in some embodiments and forcertain designs be related to w_(s) (width of strips of coated areas incertain designs) and/or w_(g) (width of the gap between strips forcertain designs). As another example, cell deformability may depend atleast in part on cell size.

In some embodiments, two or more parameters are constrainedintentionally by design. For example, as discussed herein, w_(g) may beconstrained to certain values based on r_(contact) (cell contactradius). In some embodiments, w_(s) may be fixed to equal w_(g).

Relationships between parameters may be determined experimentally. Forexample, for each w_(s) and/or w_(g), the maximum angle α_(tr) at whichcells can be made to roll on the edge with respect to direction of flowcan be determined. The density of cell adhesion molecules may alsoaffect α_(t).

E. Cell Separation and/or Collection

In certain embodiments of the invention, methods further compriseseparating at least one cell from certain cells in the population ofcells. Methods may, in some embodiments, further comprise collecting oneor more subpopulation of cells. Cell separation may facilitatediagnostic applications. For example, inventive methods may allowseparation and, as a result, detection of certain types of cells such asactivated neutrophils, circulating tumor cells, etc. Presence of suchcell types in a biological sample may be indicative of certainconditions, diseases, etc. Separated and/or collected cells may in someembodiments be used in downstream applications, for example, to culturea subpopulation of cells that is present in low quantities in a startingpopulation of cells or in a fluid. Separated and/or collected cells mayhave therapeutic value. For example, separated and/or collected stemcells may be used to regenerate tissue and/or function. Inventivemethods may be particularly suitable for certain therapeuticapplications, as cell rolling is a gentle process that does notinterfere with cell physiology.

Separation may be based on different rolling characteristics of the atleast one cell as compared to other cells from the population. Forexample, a subpopulation of cells sharing a common characteristic may beable to roll along an edge better (such as, for example, for a longerperiod of time before disengaging from the surface, with a greaterspeed, etc.) than other cells in the population. Cells in such asubpopulation, for example, may be directed along in a particulardirection using an edge that is angled with respect to the direction offluid flow, whereas cells that do not roll as easily are not divertedfrom the direction of flow.

In some embodiments, at least one cell of interest rolls along an edgeand is diverted away from the direction of flow in a certain direction.The cell may be collected at one or more collection points along and/orat the end of the trajectory/trajectories of diverted cells.

In some embodiments, a “negative” selection scheme is used in whichcells that do not roll along the edge are separated from others. Forexamples, edges may be designed to direct cells that roll along an edgeaway from a given collection point. Thus, cells that do not roll alongthe edge may be separated from others and/or collected. (See, forexample, FIG. 4A and Example 14.)

Cell separation and/or collection may, in some embodiments, befacilitated by inventive devices disclosed herein.

F. Three-Dimensional Methods

In certain embodiments of the invention, methods are adapted for use inthree dimensional systems. (See, for example, Example 15.) Such methodsare similar to those already described, except that the edge effect isachieved using a “stagnation line” of no flow rather than or in additionto an edge. Generally, such methods comprise steps of providing a threedimensional surface that is at least partially coated with an orderedlayer of cell adhesion molecules and flowing a population of cellsacross the surface. Fluid is flowed in such methods under suchconditions as to create a stagnation line of no flow that forms an angleα_(s) with the direction of fluid flow. At least one cell in thepopulation of cells being flowed comprises a cell surface moiety that isrecognized by the cell adhesion molecules, and at least one cell in thepopulation of cells rolls at least part of the time in a direction thatis α_(s) to the direction of flow.

When flowing fluid encounters a certain kind of three dimensional objecta “stagnation line” can be created. Any object and shape that createsdifferences in direction of flow can potentially be used to create astagnation line. For example, cylinders, ridges, grooves, bumps, etc.may create a stagnation line. At least part of the outer and/or exposedsurfaces of such objects and shapes may be coated with cell adhesionmolecules that may induce cell rolling.

A cell rolling on the surface will roll towards the stagnation line, andthen (under certain conditions) roll along the stagnation line andthereby follow it. Cells may roll in a direction at an angle to thedirection of fluid flow when the stagnation line is at an angle to thedirection of fluid flow. As in the case of rolling along an edge, cellsmay follow the stagnation line so long as the angle does not exceed amaximum angle α_(tr), whose value depends on the particular conditionsof the cell separation system. The stagnation line may be curveddepending on the surface under consideration and the flow field aroundthe surface. Therefore, the stagnation line can act as an edge andfacilitate cell rolling.

In certain embodiments, three dimensional surfaces comprise at least oneedge as discussed herein and may or may not include a stagnation line,as depicted in FIGS. 6A and B. For example, the edge may be on aspherical, cylindrical, etc. surface. Edges may also be along a wavysurface, along a surface with periodic bumps, etc.

II. Devices

In some aspects of the invention, devices for cell separation areprovided. In certain embodiments, such devices are designed to be usedin accordance with methods of the invention. Generally, such devicescomprise a separation flow chamber, an inlet for flowing cells into theseparation flow chamber, and an outlet for flowing cells out of theseparation flow chamber, wherein the separation flow chamber comprises asurface that is at least partially coated with an ordered layer of celladhesion molecules, and wherein the surface comprises at least one edgebetween an area coated with the ordered layer and another area that isnot coated with the ordered layer. In such devices, when cells areflowed through the inlet to the outlet, they flow at an angle α_(s) tothe direction of the at least one edge unless they interact with the atleast one edge.

Devices of the invention may comprise any of the features disclosedabove in the discussion of inventive methods.

It is to be understood that a device may include any number of inlets oroutlets as may be required for a particular application. For example, incertain embodiments, a device may further comprise an additional inletfor introducing a buffer stream free of cells into the separation flowchamber. A plurality of outlets may be useful, e.g., when it isdesirable to collect cells which are differentially separated as aresult of flowing through the separation flow chamber.

The separation flow chamber may have any shape, e.g., withoutlimitation, a square or rectangular shape.

Without wishing to be bound by any particular theory, the height of theseparation flow chamber may influence the percentage of cells beingflowed that is forced to interact with the surface. It may be desirable,in some embodiments, to limit the height such that more cells flowingthrough the separation flow chamber interact with the surface. In someembodiments of the invention, the walls defining the separation flowchamber have a height ranging from about 5 μm to about 1 μm. In variousembodiments, the height of such walls is less than about 100 μm, lessthan about 75 μm, less than about 50 μm, less than about 25 μm, or lessthan about 15 μm. The height may not be uniform through the length ofthe separation chamber. For example, the height may vary across thelength of the separation chamber in steps.

In certain embodiments, the separation flow chamber may be defined by alower partially coated surface, walls and an upper uncoated surface. Incertain embodiments, a single device may include a plurality ofseparation flow chambers each with their own inlet(s) and outlet(s).

In certain embodiments, these separation flow chambers may be separateand unable to communicate (i.e., a parallel system). Each separationflow chamber in such a device may be include the same or a differentedge design. Devices which include a plurality of separation flowchambers with the same edge design may be useful when there is a need toreplicate a separation under similar conditions (e.g., one or more testsamples and a control sample). Devices which include a plurality ofseparatin flow chambers with a different design may be useful when thereis a need to identify a design which produces optimal separation (e.g.,using different aliqouts of the same test sample).

In certain embodiments, a device may include two or more separationchambers that are in fluid communication (e.g., where the outlet from afirst separation chamber feeds into the inlet of a second separationchamber). Each separation flow chamber in such a device may be includethe same or a different edge design. It will be appreciated that suchserial set ups may be useful when, for example, it is desirable toexpose a subpopulation of cells which has been isolated by a firstseparation phase to a second separation phase (e.g., to isolatesub-subpopulations).

It will be appreciated that any combination or permutation of theaforementioned embodiments is encompassed by the present invention.

In certain embodiments of the invention, inventive devices may be usedin conjunction with other devices. For example, cells flowing out of theoutlet of one device may flow into another device. Alternatively oradditionally, devices may be fabricated such that they receive (intotheir inlets) cells flowing from another device.

In certain embodiments, devices further comprise one or more means forcollecting at least a subpopulation of cells flowed through theseparation flow chamber (e.g., one or more channels at one end of theseparation flow chamber). It will be appreciated that any of theaforementioned methods may comprise steps which make use of such meansfor collecting cells. In some embodiments, a porous filter may besituated at one end of the channel. A plurality of channels may be usedin devices for collection of subpopulations of cells. In some suchembodiments, devices further each comprise a plurality of porous filtersthat may be situated, for example, at the ends of collection channelsand/or between sequential channels.

Devices may be designed and/or built such that it is possible tovisualize collected cells easily. For example, collected cells may bevisualized by eye, using a low power microscope, using a magnifyinglens, or combinations thereof (e.g., see FIGS. 7A-7B, which shows achannel that can be visualized by eye when the channel is filled withcells). In some embodiments, visualization of collected cells in thechannel is facilitated by illumination with light. Device elements thataid visualization of cells may in some embodiments be incorporated intothe device. For example, a magnifying lens may be built into the deviceand situated such that it magnifies the collection channel.Alternatively or additionally, collected cells may be visualized withthe help of colored fluid, dyes, etc, or combinations thereof. In someembodiments, estimates of the numbers of collected cells can be obtainedwithout further processing. In some such embodiments, devices mayincorporate, for example tick marks along the collection channel suchthat the height of the column of collected cells gives an indication ofthe cell volume and/or of the cell count.

In certain embodiments, devices further comprise a means for controllingflow rate. In some embodiments, the means for controlling flow rate is asyringe pump.

In general it is to be understood that while liquid fluid flow has beenused in may of the embodiments described herein, flow of cells in aninventive method or device may be accomplished by a variety of means.Thus, while cells may be flowed in a fluid, cells may also be flowedusing capillary action. Thus, in some embodiments, cells are flowed in avacuum. In some embodiments, cells are flowed at least in part due to aforce or forces such as, for example, gravitational forces,electrokinetic forces, centrifugal forces, and combinations thereof.

EXAMPLES

The following examples describe some of the preferred modes of makingand practicing the present invention. However, it should be understoodthat these examples are for illustrative purposes only and are not meantto limit the scope of the invention. Furthermore, unless the descriptionin an Example is presented in the past tense, the text, like the rest ofthe specification, is not intended to suggest that experiments wereactually performed or data were actually obtained.

Example 1: P-Selectin-Coated Surfaces

It may be desirable in certain applications such as those describedherein to be able to control the presentation of biomolecules onsurfaces. For example, controlling the density and conformation ofbiomolecules on surfaces and enhancing stability of such coated surfacescould allow tuning of such surfaces for particular applications. Also,co-immobilization of secondary molecules may facilitate selectiveseparation of certain types of subpopulations such as mesenchymalstem/progenitor cells.

Covalent immobilization of biologically active species may beadvantageous for controlling parameters such as density, conformation,and enhanced stability. Although covalent immobilization procedures forpeptides and enzymes have been studied for decades, covalentimmobilization of large molecular weight biomolecules such as selectinspresent significant challenges. Among such challenges are increasedbinding to non-specific sites and a requirement for mild processingconditions to prevent protein inactivation.

In the present Example, P-selectin was covalently immobilized onto glasssubstrates and characteristics (such as, for example, orientation,density, and stability of P-selectin molecules, etc.) of such coatedsurfaces were examined.

Materials and Methods

Recombinant Human P-selectin/Fc chimera (P-selectin) and Human Fcantibody fragments were purchased from R&D Systems (Minneapolis, Minn.).SuperClean unmodified glass slides and SuperEpoxy® (ArrayIt®)functionalized slides were obtained from TeleChem International Inc.(Sunnyvale, Calif.). Heterobifunctional poly(ethylene glycol)(NH₂-PEG-COOH) was acquired from Nektar Therapeutics (San Carlos,Calif.). SuperAvidin™-coated microspheres with a diameter of 9.95 μmwere obtained from Bangs Laboratories. Multivalent biotinylated SialylLewis(x)-poly(acrylamide) (sLex-PAA-biotin) and a rectangularparallel-plate flow chamber with a 250 μm thick gasket were obtainedfrom Glycotech. All other chemicals used in the present Example wereobtained from Signa-Aldrich (St. Louis, Mo.).

Materials employed in the present Example were used without furtherpurification unless specified.

Preparation of Surfaces

As illustrated in FIG. 8, glass substrates containing epoxy groups(SuperEpoxy®) were first coated with 5 mg/mL of bifunctionalpoly(ethylene glycol) (M_(n) 5,000) as a spacer to provide reactivesites (carboxylic ends) to P-selectin and non-fouling surfaces.Carboxylic acid groups on the PEG linker were pre-activated using EDCand NHS, followed by reaction with P-selectin solution (5 μg/mL) at roomtemperature overnight. Resulting surfaces were washed with PBSthoroughly and stored at 4° C. for later use. P-selectin physisorbedsurfaces were also prepared on plain glass and on PEGylated glasswithout EDC/NHS activation to be used for comparison. Each step of theimmobilization process was confirmed by contact angle measurement andX-ray photoelectron spectroscopy (XPS) (Data not shown).

Results and Discussion

P-selectin-coated surfaces were prepared on epoxy-coated slides andanalyzed as discussed below.

Enhanced Functional Stability as Determined by Rolling of Microspheresand Live Cells

Avidin-coated microspheres were conjugated with multivalent biotinylatedSialyl Lewis(x)-poly(acrylamide) (sLex-PAA-biotin) and used as a cellmimic. For flow experiments using the microsphere conjugates, arectangular parallel-plate flow chamber with a 250 μm thick gasket wasplaced on the glass surfaces with P-selectin. Multivalent sLex-coatedmicrospheres (approximately 5×10⁵/mL) were perfused into the flowchamber at a shear stress of approximately 0.24 dyn/cm². Images weretaken every 5 seconds and velocities (averaged over at least 20microspheres) were calculated by measuring the displacement of eachmicrosphere in consecutive images.

Freshly prepared surfaces exhibited significantly lower microspherevelocities compared to unmodified surfaces. Microsphere conjugatestraveled on PEGylated surfaces without P-selectin at average velocitiesof 30-40 μms, which was in reasonable agreement of 57 μm/s according toGoldman's calculation (Goldman et al. 1967. “Slow viscous motion of asphere parallel to a plane wall. II Couette flow.” Chemical EngineeringScience. 22: 653-660, the entire contents of which are herebyincorporated by reference in their entirety).

After 21 days in PBS at room temperature, P-selectin covalentlyimmobilized onto epoxy glass exhibited a significantly better long termstability compared to both physisorbed P-selectin and unactivatedsurfaces (without NHS/EDC). P-selectin immobilized surfaces(pre-activated) exhibited the highest reduction in the microspherevelocity, with microspheres traveling at 40% of their velocities onPEGylated epoxy surfaces without P-selectin. P-selectin immobilized onepoxy glass untreated with EDC/NHS and P-selectin-adsorbed plain glassallowed microsphere conjugates to travel relatively faster. OnP-selectin immobilized epoxy glass untreated with EDC/NHS and theP-selectin-adsorbed plain glass, sLex-PAA-conjugated microspherestraveled at ˜85% and ˜70% respectively of their velocities on surfaceson PEGylated epoxy surfaces without P-selectin.

Neutrophil rolling interaction with the immobilized P-selectin was alsoinvestigated using a parallel-plate chamber under flow. A suspension of2.5×10⁵/mL neutrophils was perfused into the chamber at different flowrates corresponding to wall shear stresses ranging from 1 to 10 dyn/cm².A cell was classified as rolling if it rolled for >10 seconds whileremaining in the field of view (864×648 μm² using a 10× objective) andif it translated at an average velocity less than 50% of the calculatedfree stream velocity of a non-interacting cell. Control surfaces thatdid not have P-selectin (i.e., plain glass and a PEGylated epoxy glassslides) showed no cell adhesion (data not shown).

The stability of covalently immobilized P-selectin is evident from thisin vitro cell rolling assay at four different wall shear stresses (1, 3,5 and 10 dyn/cm²). The number of rolling cells significantly decreasedwith time for surfaces prepared by physisorption of P-selectin, butremained unaffected for covalently immobilized P-selectin even 28 daysafter preparation (FIGS. 10A and 10B). Specifically, at 3 dyn/cm²,rolling flux on aged surfaces with covalently immobilized P-selectin didnot exhibit a significant decrease (80.6±19.1% (mean±SEM), but fluxes onaged P-selectin adsorbed surfaces dropped to 30.1±5.2%, respectively.

Real Time Analysis of Covalent Immobilization Using SPR

To quantitatively characterize immobilization chemistries and theireffects on ligand binding, surface plasmon resonance (SPR) was employed.This flow-based SPR system offers: 1) easy surface functionalizationusing thiol chemistries due to the presence of a gold layer and 2)quantitative and real time monitoring of binding events without anymodification of analytes. Therefore, the SPR technique is usefulparticularly for determining controllability of density and orientationof P-selectin.

We developed chemistries to achieve non-fouling surfaces property and toprovide reactive sites for subsequent P-selectin immobilization usingoligo(ethylene glycol)-alkanethiols (Prochimia, Poland) on gold coatedSPR chips. Immobilization on non-fouling PEG surfaces is advantageousbecause: 1) it reduces non-specific interaction due to a high content ofPEG-OH groups, 2) it facilitates controllability of density/orientationby changing ratios between bi- and mono-functional PEG components, whichis easier and more reproducible than using different P-selectinconcentrations, and 3) it potentially enables introduction of multiplechemistries on the same surface.

Self-assembled monolayers (SAMs) were formed by soaking clean goldcoated substrates in a 100 μM solution of OEG-alkanethiols in ethanol atroom temperature overnight. The following mixtures of differentOEG-alkanethiols were used at the indicated molar ratios:OEG-COOH:OEG-OH (1:39, 1:9, 3:7, 5:5) and OEG-biotin:OEG-OH (1:9). SAMswere rinsed, dried and degassed before introduction into the SPRinstrument.

P-selectin was immobilized onto the surfaces of mixed SAMs ofOEG-COOH/OEG-OH as shown in FIG. 10A. 10 mM phosphate buffer (PB) wasfirst flowed into a chip at a flow rate of 50 μL/min for 5 min. A 1:1(v/v) mixture of excess EDC and NHS was injected to activate carboxylgroups on the SAMs for 10 min. After flowing for 5 min, P-selectin at aconcentration of 20 μg/mL in PB was injected and flowed for 7 min forimmobilization. The chip surface was then washed with PB for 5 min,followed by ethanolamine (100 mM in PB) to deactivate remaining activeester groups and to remove loosely bound P-selectin from the surface.

For P-selectin immobilization on a mixed SAM of OEG-biotin/OEG-OH,P-selectin was first biotinylated using maleimide-PEO₂-biotin (Pierce)before SPR measurement as shown in FIG. 10B. A solution of P-selectin at50 μL of 1 mg/mL P-selectin in PBS was mixed with 50 molar excessmaleimide-PEG₂-biotin solution at 4° C. overnight. The reaction mixturewas purified by 4 cycles of ultrafiltration using a 10K molecular weightcut-off membrane. Each cycle was performed at 14,000×g for 30 minutes.The mixed SAM of OEG-biotin/OEG-OH was mounted on the SPR and 10 μg/mLstreptavidin in PBS was flowed for 10 minutes to create binding sitesfor biotinylated P-selectin. P-selectin was then immobilized under thesame condition used for other mixed SAM surfaces via strongbiotin/avidin binding. Immobilization in the SPR was carried out at aflow rate of 50 μL/minute.

Stable and Tunable P-Selectin Immobilized Surfaces Characterized by SPR

To compare covalent immobilization with physisorption, some SPR channelswere used as reference channels where P-selectin was adsorbed on thesurface without EDC/NHS activation. Covalently immobilized P-selectinappeared to be stable, whereas physisorbed P-selectin was readilydetached from the surface when washed with 150 mM Tris-HCl bufferedsaline (FIG. 11). To control density of P-selectin, mixed SAMs ofOEG-COOH/OEG-OH at different ratios (using the chemistry shown in FIG.10A) were used and P-selectin was immobilized under the same conditiondescribed above. FIG. 11A shows that the amount of P-selectin waslinearly proportional to the amount of —COOH containing SAM component.Immobilization density could thus be controlled using mixed SAMs.

Orientation effect of P-selectin was also examined by comparing the twodifferent chemistries in FIG. 10A (random conformation) and FIG. 10B(oriented conformation). Because a P-selectin molecule has many aminegroups that can react with —COOH groups on the surface, conformation ofP-selectin ought to be random. In contrast, P-selectin is known topossess only one cysteine as its 766th amino acid (P-selectin used inthis study is composed of 1-771 amino acids of its natural form) on theother side of active binding sites at N terminal. For channels preparedusing both chemistries, comparable amounts of P-selectin were firstimmobilized (˜12 nm in wavelength shift), followed by flowing 20 ug/mLP-selectin antibody (eBioscience) at a flow rate of 20 μL/min. Channelswith oriented P-selectin exhibited a significantly greater bindingresponse than that from the channels with randomly immobilizedP-selectin (FIG. 11B), indicating that orientation of P-selectin wascontrolled by using thioether chemistry.

In this preliminary study on covalent immobilization, we have shown thatP-selectin can be covalently immobilized on surfaces, which providesbetter stability (as compared to physisorption) and control of densityas well as orientation of P-selectin. These results suggest that stableand tunable surfaces can be prepared using the developed chemistries.Stable and tunable surfaces may be especially advantageous forconsistency and for effective separation of cell subpopulations.

Example 2: Creation of P-Selectin Edges on Substrates

As described herein, coated surfaces with edges can facilitate cellrolling based separation. In the present Example, edges betweenP-selectin-coated areas and uncoated areas were created on glasssubstrates using silicone rubber masks.

Materials and Methods

Human P-selectin/Fc chimera (R&D Systems) was deposited on clean glassslides (SuperClean2, Telechem Inc.) using silicone gaskets as blocks toprevent parts of the glass substrate from P-selectin adsorption duringphysisorption of P-selectin. Clean silicone pieces were placed on theglass slide with their edges aligned at the desired angle to the edge ofthe glass slide. Glass slides were rinsed twice with 1×PBS, and 5 μg/mLP-selectin (in 1×PBS) was adsorbed overnight on exposed areas of theslides. Slides were then rinsed with 1×PBS, silicone pieces wereremoved, and the entire surface was blocked with 5% FBS or BSA. For someslides, BSA-FTTC (Sigma-Aldrich) was used instead of BSA or FBS forblocking in order to visualize the P-selectin coated areas.

Results and Discussion

Although covalent immobilization of P-selectin enhances surfaceproperties such as functional stability, proof-of-concept studies do notrequire long-term stability and physisorption of P-selectin on glasssubstrates is sufficient. Selective physisorption of P-selectin wasachieved using a silicone rubber mask in order to deposit P-selectin onglass substrates (FIG. 12). Use of bovine serum albumin conjugated withfluorescein isothiocyanate (BSA-FITC) during the blocking step revealedselective adsorption of BSA in the region occupied by the silicone maskas compared to the P-selectin coated region. Coated areas hadwell-defined edges, showing that the silicone mask did not leak duringthe physisorption step. Furthermore, when cells were flowed over thissubstrate, the cells selectively interacted with the region coated withP-selectin, confirming the success of the technique (FIGS. 1A, 1B, and13E).

Example 3: Directing Cell Rolling on a Substrate Comprising an AngledEdge

In this Example, the direction of cell rolling was influenced using asubstrate comprising P-selectin molecules forming a coated surface withan edge angled to the direction of fluid flow, demonstrating thepotential feasibility of separating cells using cell rolling along anangled edge.

Materials and Methods

P-selectin coated surfaces were generated as described in Example 2,with an edge between a coated region and an uncoated region. Edges wereangled at a various directions with respect to the direction of flow.

Cell and Microsphere Rolling Experiments in a Flow Chamber

Cell and microsphere rolling experiments were performed in acommercially available rectangular parallel-plate flow chamber 1 cmwide, 6 cm long, and 125 μm deep (250 μm for microspheres) (GlycotechInc.). HL-60 cells at densities of 3-5×10⁵/mL in cell culture medium ormicrospheres at densities of 10⁵/mL in 1×PBS buffer with 1% BSA wereloaded in 5 mL syringes mounted on a syringe pump (New Era Pump Systems,Inc., Farmingdale, N.Y.) for controlling the flow rate. Flow rates werevaried between 50 and 2000 μL/min, with corresponding shear stresses of0.32-12.8 dynes/cm2 (0.032 to 1.28 Pa). When cells were flowed, human Fcfragments at 5 μg/mL were added to the cell suspension before theexperiments in order to minimize interactions of the HL-60 cells withthe Fc part of the P-selectin chimera. The flow chamber was mounted onan Axiovert 200 Zeiss microscope (Carl Zeiss, Thonwood, N.Y.) and imageswere obtained using a 10× objective typically at a rate of 1 frame persecond for cell rolling and 3 frames per second for microsphere rollingfor duration of 1 to 4 minutes. Flow was laminar (Re˜0.1-3) and shearstress (τ) was calculated using plane Poiseuille flow using the equation

$\begin{matrix}{\tau = \frac{6{\mu Q}}{{wh}^{2}}} & \left( {{Eq}.\mspace{14mu} 2} \right)\end{matrix}$

where μ is the kinematic viscosity, Q is volumetric flow rate, w iswidth of the flow chamber, and h is height of the flow chamber.

Data Analysis

To facilitate data analysis, images were adjusted to the same extent forbrightness, contrast, and gamma correction and processed using ahome-made Matlab particle tracking software built around a particletracking freeware. Images were filtered using a spatial filter andbrightness threshold in order to identify cells. This step was verifiedby comparing Matlab-generated plots of cell positions with the realimage in order to ensure that there were no spurious effects duringimage processing. Cell position was further located with sub-pixelresolution by averaging over the pixel intensities to locate thecentroid of the pixels. These data for the entire set of images wereconsolidated into particle tracks listing the positions of rolling cellsat each point in time. Tracks with the cell missing in even one imagewere discarded, as were tracks corresponding to stuck cells in which thecell did not show significant displacement (30 μm over the entiresequence of images). The particle tracking program was set to athreshold of a maximum displacement of 15 μm per frame; thusfree-flowing cells were not tracked. The final result was a list ofpositions of each cell at each point of time, which could be used forvisualization and further analysis. Average cell velocities wereobtained by dividing the displacement between the start and endpositions of the track by the elapsed time. The edge of the P-selectincoated region was easily identified from the particle tracks as trackswere present only in the P-selectin coated region, and could berepresented by a line. In order to elucidate the effect of the edge oncell rolling, tracks that started within 15 μm of the edge were analyzedfor velocity and compared with tracks that started beyond 90 μm of theedge. Average velocities and velocity distributions were obtained foreach set of tracks. Microsphere data were also similarly analyzed.

Results and Discussion

We investigated the effect of a single edge of P-selectin on the motionof rolling HL-60 cells, a human myeloid cell line that expresses highlevels of P-selectin glycoprotein ligand-1 (PSGL-1) that mediates cellrolling on selectins. Rolling behavior of HL-60 cells has beencharacterized in a number of studies, including dependence on shearrate, cell rigidity and topology, and capture in a microfluidic device.HL-60 cells are robust and easy to maintain and also express levels ofPSGL-1 that are comparable to leukocytes, making them suitablecandidates for proof-of-concept studies.

Suspensions of HL-60 cells at densities of 3-5×10⁵ cells/mL were flowedover the substrates generated in Example 2 at a shear stress of about0.32 to about 12.8 dyn/cm² (0.03-1.28 Pa) using a commercially availableflow chamber. The flow chamber was rectangular with a width of 1 cm,height of either 125 μm (for cells) or 250 μm (for microspheres), andlength of 6 cm, with inlet and outlet at either end.

Only some of the cells interacted with the surface, and the remainingcells flowed through the chamber without interacting with the surface.Only those cells that interacted with the surface were analyzed.Selective rolling of HL-60 cells was observed on the P-selectin coatedregion with slower cell rolling velocities than those on the BSA-coatedregion where cells were not hindered by the formation of adhesive bonds.Typical velocities of the rolling cells in our experiments ranged fromabout 0.3 to about 1.2 μm/s for shear stresses ranging from about 0.32to about 12.8 dyn/cm², which are either comparable to or smaller thancell rolling velocities reported in other studies.

Remarkably, when rolling HL-60 cells encountered the edge of theP-selectin region, they were diverted from their original direction oftravel along the direction of the edge, demonstrating that an edge couldindeed be used to control the transport of cells through transientreceptor-ligand adhesive bonds. Under the conditions of this particularexperiment, this effect was observed only for small angles (<ca. 10-15°)between the edge and the direction of flow, and nearly all cells thatencountered the edge were deflected from their original direction oftravel and forced to follow the P-selectin edge. No edge effect wasobserved at larger edge angles; cells that encountered the edge detachedfrom the substrate and continued to flow in the direction of fluid flow.Thus, in the conditions of this particular experiment, the direction oftravel of the cells could be changed only at smaller edge angles. FIG.12 shows snapshots of cells rolling under a shear stress of 1.9 dyn/cm²(300 μL/min) with two cells highlighted, one cell in the P-selectincoated region that did not encounter the edge and another cell thatencountered the edge and was forced to travel along the edge. The cellthat encountered the edge was deflected from its direction of fluid flowand traveled at an angle of 8.6° with respect to the other cells thatdid not encounter the edge, demonstrating that a single P-selectin edgecould be used to substantially change the direction of cell rolling andhence control the transport of rolling cell.

To analyze the rolling behavior of the cells, the sequence of images wasprocessed using Matlab. Statically adhered cells were filtered out andtracks of individual cells were plotted, clearly showing the differenttravel directions of cells rolling on the edge and those rolling insidethe P-selectin region (FIG. 14A). The image acquisition rate andprocessing parameters were set so that only those cells that rolled onthe surface were tracked. Cells that did not roll moved rapidly ascompared to cells that rolled, and their large displacements per framemade it impossible to track rolling and free-flowing cellssimultaneously. Tracks are not visible in the blocked region, reflectingthat none of the cells rolled in that region. Cells rolling in theP-selectin region that encountered the edge were forced to roll on itinstead of crossing over beyond the edge, leading to an accumulation ofmoving cells being transported at an angle to the fluid flow. Thiseffect is evident in the plotted tracks (FIG. 14A) but not obvious inthe images (FIG. 14) because of statically adherent cells thataccumulated over a period of time. The effect of the P-selectin edge isvery clear when only longer cell tracks are plotted (FIG. 14B).

To elucidate the effect of the edge on cell rolling, tracks were dividedinto two sets: (a) tracks that began within a distance of 30 μm from theedge (cells that encountered the edge), and (b) tracks that began beyonda distance of 90 μm of the edge (cells that were not influenced by theedge). The direction of travel of each track was identified and plottedas a histogram (FIG. 14C), with zero angle corresponding to the meandirection of cells rolling in the P-selectin region. Direction of travelof cells that encountered the edge clearly differed from the meandirection of travel of the other cells by 4-10°. This analysis furtherconfirmed the ability of the edge to control the direction of travel ofrolling cells. Furthermore, cells near the edge rolled at an averagevelocity of approximately 1 μm/s, whereas cells away from the edgerolled at an average velocity of approximately 0.5 μm/s. These resultsdemonstrate that the P-selectin edge enabled control over the transportof rolling cells by (a) changing the direction of rolling and (b)increasing the rolling speed.

Similar experiments were performed with Sialyl Lewis(x) (sLex) coatedmicrospheres that form transient bonds with P-selectin and are used asmodels to study cell rolling. Microspheres rolled selectively on theP-selectin region with average velocity of about 3-4 μm/s at a flowspeed of about 200 μL/min, corresponding to a shear stress of about 0.33dyn/cm² (0.033 Pa). This velocity is in agreement with the inventors'previously acquired data on sLex coated microspheres rolling onP-selectin. Nevertheless, the P-selectin edge did not have a significanteffect on the direction of rolling of the microspheres. Almost allmicrospheres that encountered the edge crossed over beyond the edge andtheir direction of travel remained unchanged (FIG. 14D). Trackscorresponding to these microspheres terminated at the edge instead offollowing it. Once the microspheres detached from the edge, theycontinued flowing in the direction of fluid flow and were no longertracked due to their much higher speeds.

This observation demonstrates that two types of particles that exhibitsimilar rolling behavior on P-selectin coated surfaces can exhibitdramatically different rolling behavior on P-selectin edges. Thisremarkable difference between the rolling behavior of cells andmicrospheres at the edge is not evident in one-dimensional rolling andsuggests that the edge effect is capable of differentiating rollingparticles based on their nanomechanical properties. It is proposed,without being held to theory, that when a cell encounters the edge, anoffset between the net force acting on the cell due to fluid flow andforces exerted as the adhesive bonds dissociate cause the cell toundergo asymmetric rolling motion and follow the edge (age FIG. 15). Themoment driving the rolling motion in the direction of fluid flow may beexpected to be of the order of F_(drag)×a, where a is the radius of thecell or microsphere and F_(drag) is the fluid force acting on the cell.The asymmetric moments that cause the cell to follow the edge may beexpected to scale as F_(drag)×I_(contact), where I_(contact) is thelength scale of the contact area within which the cell or microsphereinteract with the substrate. This asymmetric moment may be expected tovanish if the area of contact is very small because the net force actingon the cell or microsphere would be aligned with the force due to theadhesive bonds, i.e., F_(drag)×I_(contact) would not be large enough tosustain this asymmetric motion but F_(drag)×a would remain relativelyunchanged. For the rigid microspheres, the contact length is limited to˜0.4-˜0.6 μm, assuming that either the bonds or linking molecules canextend by about 5 to about 10 nm. Nevertheless, cell rolling may dependon the mechanical properties of the cell, including its deformabilityand the size and extensibility of microvilli, and the contact length canbe several micrometers long for rolling HL-60 cells. Furthermore,rolling cells can extend long tethers due to extension of microvilli toseveral micrometers that effectively increases the area of interactionbetween the rolling cell and the substrate. Without wishing to be boundby any particular theory, the lack of ability to extend long tethers maybe why sLex coated microspheres selectively rolled on the P-selectinregion but did not follow the edge even when it made a small angle withthe direction of fluid flow.

This Example demonstrates that the transport of cells based on specificreceptor-ligand interactions can be controlled in a label-free manner bythe arrangement of receptors that mediate cell rolling. A single edge ofP-selectin was capable of substantially changing the trajectory ofrolling HL-60 cells with respect to the direction of fluid flow in whichthe cells would otherwise roll; at the same time, a single edge ofP-selectin affected rolling microspheres to a much lesser extent.

Example 4: Microfluidic Device

We have developed a microfluidic device that uses rolling on an edge forseparation of cells. The device was fabricated using soft lithography inPDMS (polydimethylsiloxane). First, microfluidic patterning (Delamarche,E. et al. 1997. “Patterned delivery of immunoglobulins to surfaces usingmicrofluidic networks.” Science. 276(5313): 779-78, the entire contentsof which are hereby incorporated by reference in their entirety) wasused to define lines of P-selectin on a glass or polystyrene substrate.The device was then assembled using a vacuum manifold to hold the PDMScomponent on the substrate to form a flow chamber with height rangingfrom 30 μm to 250 μm (FIG. 16). The device contained separate inlets forcell and buffer streams. This arrangement permits a parallel flow of acell stream along with a buffer stream that does not contain any cells.

As a proof-of-concept for cell separation, we examined whether theP-selectin arrangements in the device were able to nudge cells out ofthe cell stream by guiding cell rolling along the P-selectin edges. Whena stream containing HL-60 cells and microspheres and a buffer streamwere flowed through the device, some of the cells in the cell streamtethered and rolled on the P-selectin edges on the substrate. Due to theangle that the edges made with respect to the flowing cell stream, thesecells were nudged out of the cell stream and were thus separated outfrom microspheres in the original flow stream (FIG. 16B). This resultfurther demonstrates feasibility of creating a cell separation devicebased on cell rolling.

Example 5: Characterization of Mesenchymal Stem/Progenitor Cell (MSPC)Rolling on Substrates Comprising Edges

In this Example, the effect of P-selectin arrangement on the rollingdirection of MSPCs with respect to the direction of fluid flow isinvestigated. A goal of this experiment is to maximize the ability ofthe arrangements to direct trajectories of rolling cells and toinvestigate how it depends on cell properties such as size, liganddensity, and deformability.

Rolling experiments are performed in a standard commercially availableflow cell (Glycotech Inc.) using a glass slide (substrate) with selectinedges. MSPCs with cytoplasmic expression of GFP are used. Cells aremaintained in Lonza MSPC expansion media as specified by themanufacturer. To ensure MSC identity, MSCs are characterized by flowcytometry using a variety of positive and negative cell markers(Dimitroff, C. J. et al. 2001. “CD44 is a major E-selectin ligand onhuman hematopoietic progenitor cells.” Journal of Cell Biology. 153(6):1277-1286; Pittenger, M. F. 1999. “Multilineage potential of adult humanmesenchymal stem cells.” Science. 284(5411): 143-7; and Caplan, A. I.1991. “Mesenchymal stem cells.” J Orthop Res. 9(5): 641-50; the entirecontents of each of which are hereby incorporated by reference in theirentirety). Positive markers include CD90, CD 146, CD44, and CD29.Negative cell markers include two specific hematopoietic cell surfacemarkers including CD45 and CD34. Cells are incubated for 10 minutes innon-enzymatic cell dissociation solution (Sigma). After washing with PBScontaining 1% FBS and 0.05% NaN₃ (FACS buffer), cells are filtered usinga 40 μm cell strainer and incubated for 30 minutes using the followingmouse IgG,_(K) antibodies: 1) CD34 fluorescein isothiocyanate (FITC)conjugated antibody (diluted with FACS buffer), 2) CD45 FITC-conjugatedantibody, 3) CD90 FITC-conjugated antibody 4) CD146 R phycoerythrin(R-PE) conjugated antibody, 5) CD44 FITC-conjugated antibody (Abcamab30405), and CD29 PE-conjugated antibody (FAB 17781P R&D Systems),CXCR4 (R&D Systems, FAB173P).

MSPCs are also characterized for expression of P-selectin moieties usingP-selectin containing the Fc region. In addition, the ability of thecells to differentiate is verified using CFU-F and CFU-O assays. (SeeExample 7). To ensure multi-lineage differentiation potential of MSCs,adipogenic differentiation is also examined with the adipogenicSingleQuot kit from Lonza, followed by Oil Red 0 staining and FACsanalysis with FABP4 antibody (AF3150, R&D Systems). Images are acquiredusing a Nikon TE2000U microscope and analyzed using Matlab as in otherwork described herein. Specific covalent chemistry is used to immobilizeP-selectin on a substrate previously patterned with gold. This approachis chosen for the advantages of covalent chemistry over physisorption,as well for repeatability, robustness and control as compared with otherapproaches such as microfluidic patterning and microcontact printing.

Thin (˜10 nm) layers of gold are evaporated on glass slides andpatterned using standard lithography techniques. A 10 nm gold film ischosen to facilitate visibility of cell rolling through the gold film(Mrksich, M. et al. 1996. “Controlling cell attachment on contouredsurfaces with self-assembled monolayers of alkanethiolates on gold.”Proceedings of the National Academy of Sciences of the United States ofAmerica. 93(20): 10775-10778, the entire contents of which are herebyincorporated by reference in their entirety). Following goldevaporation, glass slides are treated with PEO-silane (2-(methoxy(polyethyleneoxy) propyl) trimethoxysilane, Gelest, Inc.) in order toblock adsorption of P-selectin on glass surfaces. P-selectin is thenimmobilized on gold surfaces using thiol chemistry. On gold-coatedregions, oligo(ethylene glycol) (OEG)-containing alkanethiols are firstconjugated to prepare non-fouling self assembled monolayers (SAMs). Tocontrol density and orientation of P-selectin, mono- and bi-functionalOEG-alkanethiols are employed. For example, by changing mixture ratiosbetween SH—(CH₂)_(m)—(CH₂O)_(n)—OH and eitherSH—(CH₂)_(m)—(CH₂O)_(n)—COOH or SH—(CH₂)_(m)—(CH₂O)_(n)—NH₂, density ofreactive sites (—COOH or —NH₂) that can react with P-selectin can becontrolled, resulting in controlled P-selectin density on the surface.In addition, the bifunctional OEG-alkanethiols is further reacted with alinker such as sulfo-(succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate) (sulfo-SMCC), allowingorientation control of P-selectin. Note that P-selectin has only onecysteine residue (the 766th amino acid located on the opposite side ofthe adhesive site) that contains thiol groups, which permits controlover the orientation of its adhesive amine terminus (Fujimoto T. et al.1993. “P-selectin is acylated with palmitic acid and stearic acid atcystein-766 through a thioester linkage.” Journal of BiologicalChemistry. 268(15):11394-11400). The entire surfaces are then blockedwith a 5% solution of FBS in 1×PBS.

Determination of P-Selectin Edges that Maximize Deflection of RollingCells

A basic arrangement comprising strips of selectin defined by width ofselectin strip (w_(s)), width of gap (w_(g)), and angle with respect toflow direction (α_(s)) (FIG. 5) will be used. Minimum pattern dimension(˜0.5 μm) is determined by lithography resolution. Preliminary dataindicate that, under the conditions of these particular experiments,cells are unable to be directed by an edge for large α_(s).Nevertheless, it is expected that the use of multiple edges may have asignificant effect on the trajectory of rolling cells even at largeα_(s). α_(s) will therefore be varied between about 1° and about 60°.

The area of contact of a rolling cell with the substrate and thedistance traveled by a cell before reattachment to the substrate may betwo parameters that are particularly relevant to the design of w_(s) andw_(g). Prior studies on cell rolling suggest that the area of contact istypically in the range of about 5 to about 10 μm (Dong, C. et al. 2000.“Biomechanics of cell rolling: shear flow, cell-surface adhesion, andcell deformability.” 33(1):35-43, the entire contents of which arehereby incorporated by reference in their entirety). w_(g) will bevaried between the minimum size determined by lithography resolution(˜0.5 μm) and about 10 μm, which may be an upper limit for contactdimension. Without wishing to be bound by any particular theory, thedistance traveled by a cell that detaches from an edge beforereattachment should be minimized for the most effective separation. Forw_(s)<w_(g) the fraction of substrate coated with selectin is small, andreattachment kinetics may be adversely affected. Nevertheless,increasing w_(s) beyond w_(g) gives diminishing returns, as most of thesurface becomes covered with selectin and the number of edges isreduced. An edge arrangement will therefore be set with widthw=w_(s)=w_(g). Edges will be designed such that several combinations ofα_(s) and w can be tested on a single glass slide in a singleexperiment.

Trajectories are obtained for MSPCs rolling on each arrangement fordifferent values of α_(s) and w. Width (w=gap width w_(g) and selectinwidth w_(s)) is varied as 0.5, 1, 2, 5, and 10 μm. Edge angle α_(s) isvaried as 1°, 2°, 5°, 10°, 20°, 40°, and 60°. For each width, themaximum trajectory angle α_(tr) at which cells can be made to roll withrespect to flow direction will be determined. In addition, the distancetraveled by the cells while rolling along the edge is quantified usingMatlab as in work described herein. It is expected that the maximumtrajectory angle will become independent of the width (w) if the widthis larger than the area of contact of the cell with the substrate. Theangular distribution of cell trajectories are also evaluated for eachselectin arrangement. (See the similar to the evaluation shown in FIG.14). In addition to geometry of coated areas and edges, the effect ofP-selectin density is determined by decreasing the density of P-selectinand observing its effect on the maximum trajectory angle α_(tr).

Investigation of Effects of Cell Size and Deformability on the Abilityof Substrates Comprising Edges to Direct Trajectories of Rolling Cells

Biomechanical properties of MSPCs may be expected to play a role in cellrolling and homing processes. The effect of cell size and celldeformability is investigated by controlling each parameterindependently. Without wishing to be bound by any particular theory,cell deformability may play an important role, since rolling along anedge was not observed in the case of rigid microspheres even for smallα_(s). Cell size affects the area of contact and also affects the shearforce exerted by fluid flow on the cell. In order to study the effect ofcell size, MSPCs are sorted according to cell size by flow cytometryinto 3-4 subpopulations containing more homogeneous size distributions.Cell deformability is controlled by treating the cells with cytochalasinD, which increases cell deformability. Cytochalasin D is a cellpermeable mycotoxin, which causes both the association and dissociationof actin subunits. Cytochalasin D disrupts actin filaments and inhibitsactin polymerization, resulting in disruption of cytoskeleton. Sincecytochalasin D will also interfere with the cell's ability to producepseudopod extensions to interact with the substrate, an alternative,methyl-P-cyclodextrin (MβCD) is also employed to decrease cell rigidity.MβCD is known to deplete cholesterol from cell membranes, resulting in asubstantial increase in membrane fluidity.

To determine the effect of cell size and deformability on rollingbehavior, rolling experiments are repeated for specific MSPCsubpopulations. To study the effect of cell size, maximum trajectoryangle α_(tr) is determined for 3-4 sub-populations of MSPCs sorted onthe basis of cell size. Similarly, the effect of cell deformability isdetermined by observing how cytochalasin D (or MβCD) affects the maximumα_(tr).

Example 6: Enrichment of Subpopulations of MPSC by Rolling on P-Selectin

This Example demonstrates separation of a population of cells intosubpopulations using methods and devices of the present invention.

MSPCs are separated into 3-4 subpopulations by rolling and differences(such as, for example, size and receptor density) between resultingsubpopulations is examined using flow cytometry. Separation is achievedby design and fabrication of a microfluidic device based on cell rollingcharacterization of Example 5. It is expected that the cells areseparated on the basis of size, ligand density, cell deformability, orcombinations thereof.

Device Design

Devices comprise an inlet for cell suspension, another inlet forbuffer/medium, a separation flow chamber, and several outlets (FIG. 3).Results from Example 5 are Used to Guide design of geometry of thedevice and of selectin arrangements. The cell suspension inlet width iskept to ˜30 μm, as increasing this width would likely increase theseparation distance and thereby increase the time required for cells toflow through the device. Edges of selectin are designed based on themaximum trajectory angle α_(tr) determined for each cell population inExample 5. Examples of designs that are contemplated include (a)constant edge angle, and (b) varying edge angle (FIGS. 3A and 3B).

Choice of edge and coated area designs are influenced by the sensitivityof cell trajectories to the design: if, for a design that maximizesα_(tr) for a particular subpopulation, α_(tr) is very small for othersubpopulations, a varying edge angle design may be suitable. A constantedge angle design may be suitable for other situations. The minimumlength of the flow chamber is given approximately by

$\begin{matrix}{L\text{∼}\frac{w_{inlet}N}{\tan\left( \alpha_{\max} \right)}} & {{Eq}.\mspace{14mu} 3}\end{matrix}$

where N is the desired number of fractions. For w_(inlet)=30 μm, N=4,and α_(max)=8°, the minimum length of the flow chamber is about 850 μm.Cell separation time is 5-10 min for typical rolling speeds observed inour experiments for this geometry. Since cell rolling is inherentlyslow, parallel device operation may be necessary for sufficientthroughput. For physiological shear stress of 10 dyn/cm², the cellsuspension inlet flow rate is calculated to be approximately 1 μL/min. Agoal is to separate approximately 10⁵ cells in 100 μL at a cell densityof 10⁶/mL. Ten devices are employed in parallel to separate cellsuspension at a rate of 10 μL/min (FIG. 3).

Device Fabrication

Devices are fabricated from PDMS (polydimethylsiloxane) (Sylgard 184,Dow Corning) using a standard micromolding process on a SU-8(photocurable epoxy from Microchem, Inc.) master mold. SU-8 patternswith connecting microchannels with the desired height will be fabricatedon 4″ silicon wafers using standard procedures, followed by silanetreatment to prevent PDMS sticking to the molds. A mixture of PDMS andcuring agent in the ratio 10:1 by weight is poured on the master andcured at 60° C. for 1 hour. After curing, the PDMS components will bepeeled off and access holes will be punched for inlets and outlets. Asecond layer of PDMS with connecting manifolds is similarly fabricatedand bonded to this layer using oxygen plasma treatment. Inlet and outlettubing are connected to this layer using silicone adhesive. PDMScomponents are placed on other glass slides previously coated in someareas with selectin for experiments. PDMS components and glass slidesare held together using a mechanical clamp or vacuum.

Separation of MSPCs

MSPCs are characterized for P-selectin density at different passagenumbers by FACS analysis to examine the variation of ligand expressionwith passages. P-selectin labeled with FITC using an Antibody LabelingKit (53027, Pierce) according to the manufactures protocol will be usedfor this characterization. MSPCs at a density of 10⁵-10⁶ cells/mL andbuffer or cell culture medium are flowed into the device at flow ratesof 10 μL/min and 30 to 100 μL/min (depending on device geometry),respectively. Three to five subpopulations are collected and analyzedfor (a) cell size, (b) ligand density, and (c) deformability. Cell sizeand P-selectin ligand density are characterized for each subpopulationusing BD FACS Calibur as described above. Cell deformability is assessedby forcing cells to enter narrow microfluidic channels under acontrolled pressure drop and observing the time it takes a cell to entera channel. To better understand the differences between the separatedsubpopulations, a statistical model is constructed to correlate theeffect of size, deformability, and ligand density on MSPC receptorexpression profile and on ability to form CFU-Fs (Example 7). This modelis useful to tune design parameters for enhanced separation and helpsdetermine whether cell rolling can be used to separate MSPCs based onspecific properties.

Example 7: Identification of MSPC Subpopulations with EnhancedDifferentiation and Cell Migration Potential

The present Example illustrates another potential use of cell separationsystems disclosed herein. Differentiation and migration potential ofMSPC subpopulations such as osteogenic lineage cells are examined.

It is expected that rolling-based separation of MSPCs will yieldsubpopulations that exhibit different capacities to differentiate(measured, for example, by ability to form colonies and produce bonematrix) and/or differences in migration behavior.

The osteogenic lineage provides an attractive functional assay which canbe used to effectively assess the number of progenitors within apopulation of cells. Bone nodules are each initiated by a single MSPCand are produced during de novo bone formation on a solid surface. Denovo bone formation is initiated by differentiating osteogenic cells andis marked by the presence of a cement line matrix.

The sequence of bone formation in vitro parallels that ofintramembranous bone formation during embryogenesis and endosseous woundhealing and has been demonstrated for a variety of species including ratand human and for osteogenic cells derived from human embryonic stemcells (Davies, J. E. et al. 1991. “Deposition and resorption ofcalcified matrix in vitro by rant marrow cells.” Cells and Materials.1(1):3-15; Baksh, D. et al. 2003. “Adult human bone marrow-derivedmesenchymal progenitor cells are capable of adhesion-independentsurvival and expansion.” Exp. Hematol. 31(8):723-32; and Karp et al.2006 (cited herein); the entire contents of each of which are herebyincorporated by reference in their entirety). The extracellular matrixproduced by osteogenic cells is assembled into discrete islands ofmineralized matrix called bone nodules. Through retrospective analysisof bone nodule numbers normalized to input cell numbers, one canindirectly determine the number (frequency) of recruited MSPCs(osteoprogenitors). Bone marrow contains approximately 1 in 10,000 to 1in 100,000 MSPCs per adherent cell; this number decreases with age afterreaching its peak in the mid to late 20s in humans. Although there isconsiderable interest in culture expanding MSPCs, the rate of expansionand the yields of MSPCs are inversely related to the plating density andincubation time of each passage.

The ability to enrich and/or isolate populations of osteoprogenitorcells (i.e., progenitor cells that have the capacity to form bonenodule, in some cases, after a migration event in response to stromalderived facfor-1 (SDF-1)) would serve as a proof of concept foredge-based cell rolling separation technologies.

As a control experiment, separated MSPC subpopulations are also examinedby FACS analysis to determine whether there are any differences inexpression of known MSPC markers CD45, CD90, CD44, and CD29 as describedin Example 5.

Characterization of Expression of MSPC Homing Receptor (CXCR4) onSeparated Subpopulations

MSPCs lack or have highly variable cell surface expression of many ofthe key cytokine receptors and integrins that are responsible for homingof leukocytes and hematopoetic stem cells such as the stromal derivedfactor-1 (SDF-1) receptor (CXCR4). Methods of improving trafficking andengraftment of MSCs and other cell types are a high priority forcellular therapies. Retrovirus vectors encoding homing receptors such asCXCR4 have been recently used to enhance homing and engraftment of HSCsand MSCs through increasing cell invasion in response to stromal derivedfactor-1 (SDF-1), the ligand for CXCR4, which is typically present atinflammatory sites. A more suitable alternative would be to separateMSPCs that express CXCR4 without labeling the receptors (as required inFACS sorting).

To examine whether CXCR4 positive cells exhibit different rollingbehavior, rolling of MSPCs that express CXCR4 (obtained by FACS sorting)is compared with rolling of MSPCs those that do not express CXCR4. SinceCXCR4 is not a known rolling receptor, it is anticipated that CXCR4antibodies will not affect cell rolling. If CXCR4-expressing cellsexhibit different rolling behavior, cells that express CXCR4 areseparated from those that do not express CXCR4. MSPCs are separated intosubpopulations and expression of CXCR4 in each subpopulation areexamined to evaluate whether label-free separation of CXCR4-expressingMSPCs can be achieved using cell rolling separation systems disclosedherein.

Determination of the Ability of the Isolated Subpopulations to Migratein Response to SDF-I

In addition to differences in the number of isolated osteoprogenitors,subpopulations of MSPCs may have different capacities to transmigratethrough the vascular endothelium into the target tissue. It is believedthat MSPC transmigration is mediated via interactions between the CXCR4receptor, which is expressed on a subpopulation of MSPCs, and its ligandSDF-144 which is similar to the homing mechanism of hematopoetic cells.To determine if isolated fractions exhibit different potentials toundergo a migration event followed by bone nodule production, a modifiedBoyden chamber assay is employed as previously described (Karp et al.2005). Approximately 50,000 isolated cells from each fraction will beadded to transwell filters placed into the wells of 6-well plates. Cellsare allowed to adhere for 10 hours in the presence of 15% FBS, afterwhich wells are rinsed with PBS. Following the addition of 10 or 100ng/ml of SDF-1 to the lower compartment, cells are incubated for 24hours and then cells on top of the filter are removed with a cottonswab. After rinsing the upper and lower compartments with PBS 3 times,CFU-O media is added to the upper and lower compartments. Cells areincubated for an additional 2-3 weeks with media changes every 2 or 3days. Areas containing mineralized regions are quantified usingtetracycline (Karp et al. 2005).

To determine the numbers of cells on the underside of the filters priorto switching to CFU-O media (i.e. total numbers of cells that migratedfrom each fraction in response to SDF-1), cells on the tops of somefilters are removed by scraping with a cotton swab. Whole filters arethen stained with toluidine blue and then observed under a lightmicroscope.

Quantification of the Number of Bone Nodules from SeparatedSubpopulations

The differentiation potentials of subpopulations obtained byrolling-based separation are quantified and compared to that ofsubpopulations obtained by FACS based on P-selectin ligand density. ForFACS separation, P-selectin with Fc region are used. Three to foursubpopulations of MSPCs are separated using BD FACS Calibur based onP-selectin ligand density.

The number of osteoprogenitors is quantified using a colony forming unitosteoblast (CFU-0) assay (Karp et al. 2005). To stimulatedifferentiation into osteogenic cells, media containing a-MEM and FBS issupplemented with 10⁻⁸ M dexamethasone (DEX), 50 μg/ml ascorbic acid(AA), and 5 mM Beta glycerophosphate (βgP) together with antibiotics andfungizone. Through its interaction with specific glucocorticoidreceptors, DEX has been demonstrated to stimulate osteogenicdifferentiation for progenitor cells derived from multiple tissues. AAfacilitates collagen assembly and βgP facilitiates mineralization of thecollagen. Media will be changed every 2-3 days and mineralized areas areobserved by light microscopy and by electron microscopy. Cultures aretreated either with or without osteogenic supplements to assess directedversus spontaneous differentiation into osteogenic cells, respectively.

To examine the number of colony forming unit fibroblasts (CFU-Fs) (afunctional assay for MSPCs), cells are cultured as described byCastro-Malaspina et al. 1980. “Characterization of human bone marrowfibroblast colony-forming cells (CFU-F) and their progeny.” Blood.56(2):289-301, the entire contents of which are hereby incorporated byreference in their entirety). A positive CFU-F colony is identified asan adherent colony containing more than 50 cells that is a-naphthylacetate esterase-negative and hematoxylin & eosin-positive (Baksh, D. etal. 2003). The CFU-F assay for colony forming potential provides a roughestimate for the number of MSPCs in each fraction. CFU-F in addition toCFU-O analysis provides pertinent data regarding the potential of theMSPCs in each fraction.

Examples 8-10: Systems for Separating and Detecting ActivatedNeutrophils

This year, hundreds of thousands of infants world-wide will developsepsis, a result of the body's inflammatory response to an infection,which can lead to organ failure and death. Mortality may be as high as50% for infants who are not treated, with almost half of thesepsis-related deaths occurring among infants who are born prematurely.Most sepsis detection systems rely on identification of blood plasmalevels of certain factors or blood cultures that require a centralizedlaboratory, or on clinical symptoms that are not specific. Outcomes fornegative blood cultures typically require 5 days, which is often toolate to affect therapeutic decision. A simple method to quickly detectsepsis at the point-of-care would enable required medical treatment tobe administered on time, greatly reducing infant mortality.

Recent research indicates that expression of CD64 on neutrophils is ahighly specific biomarker for neonatal sepsis that is not significantlyaffected by conditions such as fever or chemotherapy (Bhandari, V. etal. 2008. “Hematologic profile of sepsis in neonates: neutrophil CD64 asa diagnostic marker.” 121(1): 129-134 and Ng, P. 2002. “Neutrophil CD64expression: a sensitive diagnostic marker for late-onset nosocomialinfection in very low birthweight infants. Pediatric Research. 51(3):296-3-3.) Although enzyme linked immunoabsorption assays (ELISAs) andflow cytometry techniques are useful for examining CD64 levels onneutrophils, these techniques require extensive sample processing, andproper storage conditions, and are typically not amenable forpoint-of-care diagnostics.

Inventive methods for directing the trajectories of rolling cells usingasymmetric arrangements of receptors (as described herein) could beharnessed to separate and detect activated CD64 expressing neutrophilsfor rapid, label-free diagnosis of sepsis. For example, a device forcell rolling with a design such as that shown in FIG. 17A might beuseful for separating and detecting activated CD64+ neutrophils fromother cells.

We have also developed techniques for covalent immobilization ofselectins and antibodies such as CD64 that allow for arrangement andcontrol over surface density and orientation of the selectins, as wellas prolonged shelf life. This approach can be used for enhanced controlof the rolling response of sLex ligand bound microspheres and liveneutrophils compared to physisorption. The substrates provided by thepresent Example are directed toward a goal of developing a simple,stand-alone device based on cell rolling for rapid separation anddetection of neutrophils that express CD64 on a timescale of minutes,without any processing steps.

Without wishing to be bound by any particular theory, properties ofrolling cells appear to depend on cell size, receptor expression, andcell deformability. It is therefore hypothesized, again without wishingto be bound by any particular theory, that activated neutrophilsexpressing CD64 can be separated from other cell types within wholeblood. The following Examples are expected to demonstrate separation ofactivated neutrophils from non-activated neutrophils with highspecificity. These methods can then be used to effectively separate CD64neutrophils from whole blood.

Example 8: Development of P-Selectin and Anti-CD64 AntibodyCo-Immobilized Substrates

In the present Example, substrates useful for detecting activatedneutrophils are developed. Such substrates comprising a mixture of celladhesion molecules (in this Example, P-selectin) and antibodies for amarker expressed by activated neutrophils (in this Example, CD64).

Coating Surfaces

P-selectin and anti-CD64 antibody will be coated onto surfaces such thatedges are created between coated areas and uncoated areas usingmicrofluidic patterning (FIG. 18). In this technique, microfluidicchannels in polydimethylsiloxane (PDMS) are reversibly bonded to a glassslide, and the desired receptor solution is flowed through themicrofluidic channel for immobilization. Microfluidic channels areprepared using SU-8 master mold and soft lithography techniques (Duffy,D. C. et al. “Rapid prototyping of microfluidic systems inpoly(dimethylsiloxane).” Analytical Chemistry. 70(23): 4974-4984, theentire contents of which are hereby incorporated by reference in theirentirety).

Approximately 50 μm thick SU-8 photoresists are drawn into 50 μm widelines that define the microchannels on a four inch silicon wafer. Afterprocessing, the mold is baked at approximately 150° C. for 15 min tosmoothen the edges of SU-8. The SU-8 mold is then placed in a desiccatorwith a few drops oftridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (United ChemicalTechnologies, Bristol, Pa.) to aid in the future removal of PDMS.Monomer and curing agent are mixed in a 10:1 ratio, poured over themold, degassed, cured at 90° C. for 30 minutes, and then removed fromthe mold. Inlet and outlet holes are drilled and the PDMS microchannelsare placed on the glass substrate, forming closed microchannels throughwhich solutions can be flowed using syringe pumps. We have alreadydemonstrated microfluidic creation of P-selectin edges on glass byphysisorption (FIG. 18). For better control over the surface densitiesof P-selectin and anti-CD64 mAb, we are further developing thistechnique for covalent co-immobilization of the two receptors.

Immobilization Scheme

Epoxy chemistry is used to covalently immobilize receptors on glasssubstrates. Epoxy-functionalized glass slides is obtained from ArrayltInc. and used directly for covalent immobilization without furthertreatment. The PDMS microfluidic component is placed on the epoxy slideand P-selectin and/or anti-CD64 mAb solutions in PBS buffer is flowedthrough the microfluidic channels. After immobilization, the PDMScomponent is peeled off and the entire surface is blocked with 5 mg/mLBSA for 1 hour.

Density Control of Co-Immobilized P-Selectin and Anti-CD64 mAb

Control of surface densities of P-selectin and anti-CD64 mAb may beimportant for developing an optimized cell separation device. To testdifferent densities of P-selectin and anti-CD64 mAb, theirconcentrations are varied in the solution during microfluidicpatterning. Initial experiments use different ratios ofP-selectin:anti-CD64 mAb concentrations of 1:1, 10:1, and 20:1 (withP-selectin concentrations kept at about 5 μg/mL). Total densities arevaried by varying the immobilization time of the two receptors (about 5minutes and about 1 hour) at the same P-selectin concentration to obtainlow and high surface densities at the three ratios. After arrangement ofthe receptors, surfaces are blocked with 5 mg/mL BSA for 1 hour.Surfaces are characterized qualitatively using fluorescence measurementsand quantitatively for surface density of P-selectin and anti-CD64 mAbusing a radio-labeling technique as described below.

Fluorescence Characterization of P-Selectin

Biotinylated sialyl Lewis(x) (sLex) is obtained from Glycotech andincubated with Alexa 488 streptavidin (Invitrogen, Inc.) in a molarratio of 1:1 at a streptavidin concentration of 1 mg/mL in PBS. sLex isa saccharide that binds to P-selectin and is used for surface coating ofmicrospheres that mimic cell rolling (Hong, S. et al. 2007. “Covalentimmobilization of P-selectin enhances cell rolling.” Langmuir.23(24):12261-12268, the entire contents of which are hereby incorporatedby reference). For characterization of anti-CD64 mAb immobilization,recombinant human Fey receptor I (CD64) consisting of the extracellulardomain of the Fey receptor is obtained from R&D Systems and labeled withAlexa 647 using a protein labeling kit (Invitrogen, Inc). Surfaces areincubated with 10 μg/mL solution of the streptavidin-sLex conjugate and10 μg/mL Fey receptor overnight at 4° C. After incubation, slides arerinsed twice with 1×PBS for 10 minutes and imaged under a Nikon TE2000-Uinverted epifluorescence microscope equipped with an Andor 885 camerafor imaging. Fluorescence images using filters for Alexa 488 and forAlexa 647 are acquired under identical conditions and their intensitiesquantified to verify control over immobilization of P-selectin andAnti-CD64 mAb.

Site Density Measurements Using Radio-Labeling with Iodine (¹²⁵I)

Site densities of substrate-bound ligands are measured by radioactivitythrough iodinating (¹²⁵I) anti-CD64 antibody or P-selectin prior toexposure on the substrate. Radio-iodination of the ligands is performedusing the IODO-GEN® Iodination Reagent kit (Piercenet, Ill.) accordingto the manufacturer's protocol. Antibodies are purified prior toiodination using protein A beads (Piercenet, Ill.) and then iodinatedusing tubes coated with iodogen (typically a ratio of 10 μg or less ofthe IODO-GEN® Reagent per 100 μg of antibody). To prevent oxidation ofthe ligands, 500 μCi of carrier-free Na¹²⁵I is first added to theIODO-GEN tubes and incubated for 10-15 minutes with agitation followedby addition of the ligand solution (100 μg of purified antibody sampledissolved in 100 μL PBS).

The sample is removed from the reaction tubes to terminate theiodination of the sample by adding tyrosine-like molecules such as4-hydroxyphenyl propionic acid or 4-hydroxyphenyl acetic acid (˜50 μL of10 mg/mL), which binds to active radioiodide. Next, the radio-iodinatedligand fraction is purified and separated from iodotyrosine andunlabelled ligands by passing through a gel filtration column (providedin the kit form Pierce). The radio-iodinated ligands are stored inbuffer at 4° C. and are used fresh for each analysis to minimize loss ofthe radioactivity.

Radioactivity of the labeled ligands per unit mass (specificradioactivity) is measured with the gamma counter as μCi/mmol. For agiven mass or concentration of antibody (as measured, for example, by aprotein bicinchoninic acid (BCA) assay), it is possible to measure theradioactivity; from the molecular weight of the antibody, it is possibleto calculate the amount of radioactivity per unit mass or in terms perantibody.

To determine site densities of immobilized P-selectin or anti-CD64,¹²⁵I-labeled ligands are covalently immobilized as described above.P-selectin and anti-CD64 mAb site densities are analyzed separately foreach surface. Surfaces are then washed three times with PBS, 1.5 mMCa²⁺, 0.1% Triton X-100. Bound ligand are removed by 0.1 M NaOH, andradioactivity are measured using a gamma counter.

Example 9: Characterization of Neutrophils on Co-Immobilized Substrates

In this Example, the effect of edges (generated by areas coated withP-selectin and anti-CD64 mAb) on the rolling direction of neutrophilswith respect to the direction of fluid flow are investigated. A goal ofthis study is to maximize the ability of arrangements to directtrajectories of activated neutrophils as compared to non-activatedneutrophils by varying P-selectin and anti-CD64 mAb surface densitiesand edge angles. This study facilitates the design of a device for cellseparation and helps determine relative sensitivities of the separationtechnique to neutrophil activation.

Rolling experiments are performed in a standard commercially availableflow cell (Glycotech Inc.) using a glass slide (substrate) withco-immobilized arrangements of P-selectin and anti-CD64 mAb. Neutrophilsobtained from AllCells Inc. are kept in sterile Hanks' balanced saltsolution containing 0.5% human serum albumin, 2 mM Ca²⁺, and 10 mM HEPESat pH 7.4 until flow experiments are conducted as previously described(Hong et al. 2007). To activate neutrophils, 5×10⁶ cells/mL in HBSS areincubated for 30 minutes at 37° C. with 2 nM TNF-α (pre-dissolved in PBScontaining 4 mg/ml BSA). Neutrophils are flowed over the glass slide ata shear stress of about 1 dyn/cm², which is within the range ofphysiological shear stress. Images are acquired using a Nikon TE2000Umicroscope and analyzed using Matlab as in our present work.

Quantification of Site Density of CD64 Expression on ActivatedNeutrophils

To determine the site density of CD64 on the primary human neutrophilsurface, flow cytometry is performed using microbeads of specificantibody binding capacity (ABC) (Quantum Simply Cellular kit;Sigma-Aldrich) (FIG. 19). When microbeads are labeled with a specificantibody, they can serve as a set of standards to calibrate thefluorescence scale of the flow cytometer in units of ABC (number ofAntibodies Bound per Cell or microbead). The Quantum Simply Cellular kitis a mixture of four highly uniform microbead populations of knownantibody binding capacities. The microbeads are labeled under the sameconditions as cells and with an equal amount of the test antibody as theexperimental samples. Median values of the fluorescence intensity of thefour peaks corresponding to the four microbead populations are used toconstruct a calibration curve.

Approximately 500 uL of each of the 4 IgG labeled beads (with varyingdensities) is added to 50 μL of the cell medium and vortexed.Approximately 10 μg/mL of anti-biotin-FITC antibody (anti-CD64, Abeam,ab34224) is incubated in the dark for 30 minutes with each of thelabeled bead samples or with the cell suspension. About 2 mL of cellsuspension solution is added and centrifuged at 2500×g for 5 minutes.Samples be rinsed 2 times (centrifuged at 2500×g for 5 minutes) and thenplaced into 500 μL of the same solution as the cells to be analyzed.Microspheres and cells are analyzed using flow cytometry. A flow rate ofapproximately 100-200 events per second is used with approximately 1000events collected per bead population. Blank beads without stain serve asa negative control.

Using a forward scatter versus side scatter dot plot, a live gate aroundthe singlet population of microspheres is constructed and the peak(median) histogram channels of each of the five populations ofmicrospheres are determined in the corresponding fluorescent channel forentry into the QuickCal® spreadsheet (software available atwww.bangslabs.com). Unstained cells are used as a negative control andrun at the same instrument settings as the bead standards. The ABC valueof the unstained cell sample is subtracted from the ABC values of thestained cell samples.

QuickCal® is used to generate a calibration curve, determine theinstrument detection threshold, and quantify the ABC values of unknownsamples. To establish a calibration curve, the ABC (y-axis) is plottedversus the peak channel (x-axis) for each of the 4 antibody-bindingmicrospheres. For linear fluorescence, a log-log plot of the data shouldgive a 45° line. For ABC detection threshold determination, aftercompleting the ABC calibration procedure and plotting the calibrationcurve, the peak (median) channel of the reference blank is recorded (insome experiments the unstained cell sample is used as the referenceblank). The calibration plot is then used to determine the ABC valueassociated with the fluorescence of the reference blank (or unstainedcells). This is the ABC detection threshold of the instrument at theseinstrument settings. The detection threshold is the lowest number of ABCunits detectable above instrument noise. For ABC quantitation ofsamples, after completing the ABC calibration procedure described aboveand plotting the calibration curve, the unknown cell samples aredetermined using the flow cytometer (with exactly the same instrumentsettings as used for ABC calibration). The sample's peak (median orgeonetric mean) channel value for each population will be determined andthe calibration plot used to determine the ABC value that corresponds toeach of the sample's peak channels. The ABC value of the unstained cellsample is subtracted from the ABC values of the stained cell samples.The cell area is determined by examining the diameter of 10 cells insuspension at 40× and used to calculate the CD64 site density.

Determining Optimal Surface Densities of P-Selectin and Anti-CD64 mAb

Rolling of activated and non-activated neutrophils are firstcharacterized to maximize differences in their rolling behavior on aplain surface comprising co-immobilized P-selectin and anti-CD64 mAbwithout any angled edges. Cell suspensions at a density of approximately5×10⁴ cells/mL are flowed over the receptor-coated substrate in a flowchamber using a syringe pump at a shear rate of about 1 dyn/cm². Forthis study, surfaces comprising edges between coated areas and uncoatedareas are not used since the goal is to analyze rolling behavior withoutedge effects. Cell rolling is studied separately for activated andnon-activated neutrophils and analyzed using Matlab for the number ofrolling cells, number of stuck cells, and rolling velocities.

Without wishing to be bound by any particular theory, it is predictedthat differences in rolling behavior between activated and non-activatedcells may increase as the surface density of anti-CD64 mAb is increased,affecting the rolling velocity and number of cells interacting with thesurface. Nevertheless, the number of statically adherent cells may alsoincrease at higher surface densities of anti-CD64 mAb. An intermediatevalue may be optimal for separation of activated neutrophils fromnon-activated neutrophils. The number and velocity of rolling cells aretherefore be quantified as well as the number of statically adherentcells for different surface densities of P-selectin and anti-CD64 mAb(as described in Example 8). This study should identify a surfacepreparation with densities of P-selectin and anti-CD64 mAb that maximizedifferences in rolling velocity between activated and non-activatedcells while minimizing number of statically adherent cells.

Determining Edge Angle to Maximize Difference Between Trajectories ofActivated and Non-Activated Neutrophils

After identifying the P-selectin and anti-CD64 mAb surface densities, anoptimal edge angle (α_(s)) is identified to maximize the separation ofactivated and non-activated neutrophils. A design comprising stripes ofselectin/mAb defined by width of selectin strip (w) and angle withrespect to flow direction (α_(s)) (FIG. 5) is used. w is fixed atapproximately 50 μm and the edge angle (α_(s)) that maximizes differencebetween direction of travel of activated and non-activated neutrophilsis determined.

To most closely match conditions in the final device, a microfluidicflow chamber with a channel height of 15 μm as in the proposed devicedesign (described in Example 10) is used instead of the commerciallyavailable flow chamber for this set of experiments. Trajectories areobtained for activated and non-activated neutrophils rolling onarrangements for different values of α_(s) ranging from 5°, 10°, 20°,30°, 40°, and 50°. Matlab analysis of cell tracks is carried out todetermine (a) fraction of rolling cells that continue into free streamwhen they encounter an edge, (b) fraction of rolling cells that startfollowing an edge, (c) path traveled by each cell while following anedge before detachment, and (d) velocity of rolling of each cell. Thesedata can be easily extracted with a little modification to the Matlabprogram we are currently using. The net average deflection perpendicularto the flow direction that a rolling cell can undergo due to theP-selectin and anti-CD64 mAb arrangements is calculated as the averagepath length multiplied by sin(α_(s)). The edge angle (α_(s)) thatmaximizes the difference between the rolling of activated andnon-activated neutrophils is identified.

To verify that the difference in deflection is indeed due to CD64,control experiments using only P-selectin are performed. If the rollingbehavior of activated and non-activated neutrophils is similar onP-selectin coated surfaces, the difference may be attributed to CD64expression on the activated neutrophils. Furthermore, the number ofnon-activated and activated neutrophils that adhere to a surface coatedonly with anti-CD64 mAb is quantified. This is expected to confirm thatthe altered rolling behavior of the activated neutrophils is indeed dueto CD64 expression.

Example 10: Microfluidic Device to Distinguish CD64⁺ ActivatedNeutrophils from Non-Activated Neutrophils

The present Example is directed to providing a device that candistinguish between activated and non-activated neutrophils.

After identifying receptor densities and arrangements that maximizedifference between trajectories of activated and non-activatedneutrophils, microfluidic devices to distinguish between the two cellstates are fabricated. Studies of neutrophil CD64 expression have shownthat CD64 expression of neutrophils exhibits a single Gaussiandistribution; furthermore, CD64 expression increases several fold by afactor of 10 or more as entire distribution shifts during sepsis (Davis,B. H. et al. 2006. “Neutrophil CD64 is an improved indicator ofinfection of sepsis in emergency department patients.” Archives ofPathology & Laboratory Medicine. 130(5):654-661.). Therefore, a devicethat distinguishes between CD64⁺ or CD64⁻ neutrophils should be usefulto detect conditions of sepsis.

For separation of cells using rolling, two major modifications tocommercially available flow chambers would be advantageous. Commerciallyavailable flow chambers that are typically used in cell rolling studies(Hong et al. 2007) have heights in the range of about 125 μm or larger,which results in most cells just flowing through the chamber withoutever encountering the receptor-coated surface or edge. Such flowchambers have only one inlet and one outlet, which is not useful forseparation of cells.

In contemplated devices of the present Example, the height of thechannels is decreased to about 15 μm to promote interaction between thecells and the surface. Furthermore, two inlets (cell and buffer) and twooutlets is incorporated for separated cells. Decreasing the dimensionsof the flow chamber may adversely affect the throughput of the device.On the other hand, the high density of neutrophils in blood requiresanalysis of very minute sample volumes and therefore likely avoidsissues with throughput. In other applications where higher throughput isnecessary, these devices could be manufactured to operate in parallel.Indeed, the technology to fabricate thousands of integrated chambers ina single device (Thorsen, T. et al. 2002. “Microfluidic large-scaleintegration.” Science. 298(5593), 580-584, the contents of which arehereby incorporated by reference in their entirety) has already beencommercialized (Fluidigm, Inc.) and is in routine use in severalacademic laboratories worldwide.

Device Design

The device comprises an inlet for cell suspension, another inlet forbuffer, a separation flow chamber, and two outlets (FIG. 20). The deviceis fabricated from PDMS (polydimcthylsiloxane) (Sylgard 184, DowCorning) using a standard micromolding process on a SU-8 (photocurableepoxy from Microchem, Inc.) (Duffy et al. 1998). If necessary,supporting posts or hard backing using a glass slide are used to preventcollapse of the microchannel. P-selectin and anti-CD64 mAb areimmobilized separately on a glass slide using microfluidic patterning.The device will be assembled using a vacuum manifold to hold the PDMScomponent against the glass substrate with receptors.

Results from Example 9 are used to guide design of the device, forexample, in terms of geometry, receptor densities, and edge angle. Thecell suspension inlet width is kept to ˜20 μm, since increasing thiswidth increases the separation distance and thereby increases the timerequired for cells to flow through the device. It is anticipated thatfor deflection angles of the order of 10°, a flow chamber with length onthe order of 1-10 mm may be needed for separation, giving cellflow-through time in the range of 1-10 min. For physiological shearstress of about 1 dyn/cm², the cell suspension inlet flow rate is slow,on the order of about 1 μL/minute. Nevertheless, with the very highdensity of neutrophils in blood, small amounts of blood (˜1-10 μL)should be sufficient for collection and quantification of separatedcells. Thus, a single device may be used to separate and quantify CD64⁺neutrophils on a timescale of minutes.

Using a long separation chamber, the lateral distribution of the flux ofcells at different positions along the separation channel is determinedindependently for CD64⁺ and CD64⁻ neutrophils under the same flow andsurface design conditions. This information will facilitate designingthe device outlets such that the CD64⁺ neutrophils are divertedselectively into outlet A, while CD64⁻ and non-rolling cells flow intooutlet B (FIG. 20).

Cell Separation

Approximately 10 μL of neutrophil suspension at a density of 5×10⁴cells/mL (typical of physiological density in blood) and buffer areflowed into the device at shear stress of 1 dyn/cm². Fractions ofseparated cells in each of the outlets are collected and quantified forrelative distribution of cells in each outlet (FIG. 20). Volumescollected are measured using a pipette and added to 96 well plates.Cells are allowed to settle at the bottom of the well and manuallycounted under a microscope. For each separation experiment, the finaloutput (φ) is the relative ratio of the number of cells in outlet A(n_(A)) as compared to the number of cells in outlet B (n_(B)):

$\begin{matrix}{\varphi = \frac{n_{A}}{n_{B}}} & \left( {{Eq}.\mspace{14mu} 4} \right)\end{matrix}$

Separate experiments are performed for activated neutrophils (mimickingthe sepsis condition) and non-activated neutrophils (normal condition).A significant difference in the relative distribution (φ) betweenactivated and non-activated neutrophils indicates successfulidentification of activated neutrophils from non-activated neutrophils.

An objective criterion for φ is established based on these preliminaryresults to distinguish between non-activated and activated cells as acutoff ratio φ_(C). If φ<φ_(C), then the result is FALSE for presence ofCD64+ neutrophils, else it is TRUE. Several separation experiments (>10each for activated and non-activated neutrophils) are then carried outwith this objective criterion to quantify the specificity andsensitivity of the technique.

To determine the impact of CD64 site density on separation efficiency,we are treating primary human neutrophils with approximately 0.5 nM, 2nM, or 5 nM TNF-α, determining their site density using the bead methoddescribed in Example 9, and determining separation efficiency describedherein. This study is expected to yield the sensitivity of the devicefor various levels of CD64 expression on neutrophils.

Example 11: Development of Surfaces for Selective Rolling of HT29 CellsAlong an Edge

In this Example, surfaces for separation of cancer cells from leukocytesare developed. Such cell rolling-mediated separation of cancer cells maybe useful in diagnostic applications. HT29 is a well-established cellline that interacts with E-selectin and has been used as a circulatingtumor cell model for metastasis. HL60 cells is a myeloid cell line thatis used as a model for leukocyte cell rolling. This Example intends todemonstrate that HT29 cell can be selectively separated from HL60 cellsusing cell rolling based separation systems of the present invention.

Surfaces comprising edges between coated and uncoated areas aredeveloped with coimmobolized E-selectin and epCAM Ab to enableseparation of HT29 cells by rolling. Covalent chemistry is used toco-immobilize E-selectin and epCAM mAb (R&D Systems).

Covalent Immobilization of E-Selectin and epCAM mAb with ControlledDensity on a Glass Substrate

Epoxy chemistry is used to covalently immobilize the receptors on glasssubstrates. Epoxy-functionalized glass slides is obtained from ArrayltInc. and used directly for covalent immobilization without furthertreatment. E-selectin and epCAm mAb is arranged on surfaces usingmicrofluidic patterning (Delamarche et al. 1997). In this technique,microfluidic channels in polydimethylsiloxane (PDMS) is reversiblybonded to the glass slide, and the desired receptor solution comprisingan appropriate mixture of E-selectin and epCAM mAb is flowed through themicrofluidic channel for immobilization. After immobilization, the PDMScomponent is peeled off and the entire surface is blocked with 5 mg/mLBSA for 1 hour.

Control of surface densities of E-selectin and epCAM mAb may beimportant for developing an optimized cell separation device. To testdifferent densities of E-selectin and epCAM mAb, their concentrationsare varied in the solution during microfluidic patterning. Initialexperiments use different ratios of E-selectin:epCAM mAb concentrationsof 1:1, 10:1, and 20:1 (with E-selectin concentrations kept at about 5μg/mL). Surfaces are characterized for surface density of E-selectin andepCAM mAb using a radio-labeling technique as described below.

Site density is measured using radio-labeling with iodine (¹²⁵I) andusing methods similar to those described in Example 8. Site density ofepCAM expression in HT29 cells is quantified using flow cytometry asdescribed in Example 9.

Characterization of HT29 and HL60 Rolling on Co-Immobilized Substrates

The effect of edges between E-selectin and epCAM mAb coated areas anduncoated on the rolling direction of HT29 and HL60 cells with respect tothe direction of fluid flow is investigated. A goal of this study is tomaximize the ability of the edges to direct trajectories of HT29 cellsversus HL60 cells by varying E-selectin and epCAM mAb surface densitiesand edge angles. This study is expected to facilitate designing a devicefor cell separation and help determine relative sensitivities of theseparation technique to neutrophil activation. Rolling experiments areperformed in a microfluidic cell rolling devices we developed using PDMSmicrofabrication. The cells are flowed over the glass slide at shearstress of 1 dyn/cm², which is within the range of physiological shearstress. Images are acquired using a Nikon TE2000U microscope andanalyzed using Matlab. Optimal surface densities of E-selectin and epCAMmAb are determined using methods similar to those described in Example9.

Determining Edge Angle to Maximize Differences Between Trajectories ofHT29 and HL60 Cells

After identifying the E-selectin and epCAM mAb surface densities, theoptimal edge angle (α_(s)) to maximize separation of HT29 and HL60 cellsis determined. A design comprising stripes of selectin/mAb defined bywidth of selectin strip (w) and angle with respect to flow direction(α_(s)) (FIG. 5) is used. The width of selectin strips are fixed at w=10μm (slightly larger than the adhesion area of a rolling cell) and theedge angle (α_(s)) that maximizes difference between direction of travelof HT29 (circulating tumor cells) and HL60 (leukocytes) is determined.

Cell rolling trajectories are obtained for HT29 and HL60 cells using amicrofluidic flow chamber as described in Examples 9 and 10.

Example 12: Microfluidic Device to Separate HT29 and HL60 Cells

In this Example, optimized surfaces for selective rolling of HT29 cells(developed in Example 11) are incorporated into microfluidic devices forseparating HT29 cells from HL60 cells. Such devices may be modified forother cell separation devices that may have diagnostic applications. Forexample, they may be modified for separating and allowing detection ofcirculating tumor cells from blood or blood products. (See Example 13).

Device Design

Devices comprise an inlet for cell suspension, another inlet for buffer,a separation flow chamber, and two outlets (similar to the deviceschematic depicted in FIG. 20) fabricated from PDMS using a standardmicromolding process (Duffy et al. 1998). If necessary, supporting postsor hard backing using a glass slide are used to prevent collapse of themicrochannel. E-selectin and epCAM mAb are immobilized separately onglass slides using microfluidic patterning as described in Example 11.Devices are assembled using a vacuum manifold to hold PDMS componentsagainst the glass substrates with receptors.

Results from Example 11 are used to guide design of the device, forexample, geometry, receptor densities, and edge angle. The cellsuspension inlet width is kept to ˜20 μm, since increasing this widthincreases the separation distance and thereby likely increases the timerequired for cells to flow through the device. It is anticipated thatfor deflection angles of the order of 10°, a flow chamber with length ofthe order of 1-10 mm may be needed for separation, giving cellflow-through time in the range of 1-10 min. For physiological shearstress of about 10 dyn/cm², the cell suspension inlet flow rate is onthe order of 1 μL/min. Using a long separation chamber, the lateraldistribution of the flux of cells at different positions along theseparation channel is determined independently for HT29 and HL60 cellsunder the same flow and surface arrangement conditions. This informationwill be used to design device outlets such that HT29 cells are divertedselectively into one outlet, while HL60 cells flow into the otheroutlet. (See FIG. 20 for a similar device schematic.)

Separation Throughput

A single device may, for example, be capable of cell separation at therate of approximately 1 μL/min. Such a rate may be sufficient forinitial development and testing of the device, but inadequate forprocessing of large sample volumes. Nevertheless, it is possible toconstruct multiple separation chambers that operate in parallel due tothe inherent simplicity of the device geometry. With an estimatedfootprint of 10 mm², a single device could accommodate 100 chambers inparallel enabling a throughput of 100 μL/min. (This throughput ratecould scale up to multiple mL/min in a larger device). These devices canbe fabricated in multilayer PDMS and attached to the same substrate withreceptor arrangements.

Cell Separation

A cell suspension comprising HL60 cells at a density of 10⁵ cells/mL(typical of physiological leukocyte density in blood) spiked with HT29cells is flowed into the device at shear stress of 1 dyn/cm². HT29 cellsare stained with calcein for subsequent analysis. Concentrations of HT29cells are varied from 1 to 10³ cells/mL to span the range of clinicallyrelevant concentrations (Nagrath, S. et al. 2007. “Isolation of rarecirculating tumor cells in cancer patients by microchip technology.”Nature. 450(7173):1235-U10, the entire contents of which are herebyincorporated by reference in their entirety). Fractions of separatedcells in each of the outlets are collected and quantified for relativedistribution of cells in each outlet by flow cytometry. Selectivity ofthe separation process is quantified as the fraction of HT29 cells inthe separated sample. Yield is quantified as the fraction of HT29 cellsthat are separated compared to the total number of HT29 cells in thesample. Selectivity and yield are quantified as a function of the HT29spiked concentration in the cell suspension.

Example 13: Separation or Circulating Tumor Cells (CTCs) from WholeBlood Samples of Cancer Patients

In this Example, systems for separating circulating tumor cells (CTCs)from bodily fluids such as blood samples are developed, building onresults from Examples 11-12.

Viable clinical samples of blood from late stage colon cancer patientswith metastasis are obtained. Anticipated levels of CTC in such samplesare high. Cell rolling experiments are performed on whole blood sampleswith epCAM and E-selectin co-immobilized substrates to separate CTCsfrom leukocytes without pre-labeling or processing of samples. Bloodsamples with a high fraction of CTCs are analyzed by flow cytometry(using a with a BD FACS Calibur flow cytometer) using epCAM mAb toquantify the density of CTCs in blood. Approximately 100 μL-1 mL of thesame sample of blood (depending on device throughput) are flowed throughthe device for separation using the same surface arrangements and flowconditions as in Example 12.

The resulting fractions are analyzed by flow cytometry to quantify thenumber of CTCs separated from blood. An iterative approach may be usedto facilitate characterization of rolling CTCs, as rolling of CTCscannot be directly characterized due to their small number compared toother cells. If separation is not obtained with the surfaces and flowrates obtained in Example 12, the edge angle is increased just beyondthe angle at which CTCs can roll along the receptor edge. If CTCs arenot detected under these conditions in the human blood samples, HT29cells are spiked in blood and the lowest concentration at which they canbe detected and separated are determined. To enhance the ability totrack HT29 cell separation, HT29 cells are pre-labeled with CellTrackerGreen CMFDA (Molecular probes).

Example 14: Additional Arrangements for Use in Cell Separation Systems

In addition to the arrangements discussed in Example 6 and depicted inFIG. 3, a variety of other arrangements may be used to achieve cellseparation. Some such arrangements are depicted in FIGS. 2 and 4.“Negative” selection of rolling cells may be achieved, for example, byusing edges to divert undesired cells. (See, for example, FIG. 4A.) Insuch cell separation schemes, desired cell populations are not divertedby edges and move in the direction of fluid flow. Cells that are notdesired roll along edges designed to induce rolling of the cell type(s)of the undesired cells.

Arrangements may be designed to separate cells into single files, whichmay be useful for certain downstream analyses and/or applications. (See,for example, FIG. 4B.) Alternatively or additionally, arrangements mayincorporate elements designed to capture cells in certain locations onsurfaces, as depicted in FIG. 4C. For example, elements may be physicalstructures that impede cells from flowing in the direction of flow. Suchphysical elements include microwells, which could be depressions in thesurface where cells may become trapped. In some embodiments, patches ofadhesive ligands (such as, for example, antibodies) that facilitate cellimmobilization, etc. are used to trap cells. Arrangements mayincorporate adhesive areas leading to edges to enable cells to rollbefore encountering the edge. (See, for example, FIG. 4D).

Net displacement of two cell types may be achieved by using arrangementscomprising at least two edges that form different angles to thedirection of flow. (Sec, for example, FIG. 2.) A first edge may make anangle such that both types of cells roll along it. A second edge maymake a larger angle or have a different receptor composition such thatonly one cell type (whose trajectory is indicated by dashed lines inFIG. 2) can roll along that edge. The repeating pattern depicted in FIG.2 can be spatially varied by changing, for example, the second edgegradually over a large area; such a change may facilitate separation ofa particular cell type.

As illustrated by this Example and by other arrangements describedherein, arrangements may have any of a diverse number of geometricdesigns and may or may not incorporate certain elements (such as, forexample, microwells, patches of adhesive ligands, etc.) depending on theapplication.

Example 15: Three-Dimensional (3D) Devices for Cell Separation

In this Example, three-dimensional (3D) devices for cell separation willbe provided. As discussed and exemplified herein, in two-dimensionalsystems of the invention, an edge between areas coated with celladhesion molecules (such as, for example, P-selectin and/or E-selectin)and uncoated areas facilitates cell rolling. Cells roll along the edgeand are directed along a particular direction at an angle to thedirection of fluid flow.

On three-dimensional surfaces, an effect similar to the edge effect canoccur. A schematic of a three-dimensional device is depicted in FIG. 21.Streamlines indicating fluid flow are depicted by arrows. When flowingfluid encounters an object such as, for example, a cylinder (FIG. 21 A)or a ridge (FIG. 21B), a “stagnation line” can be created. In such 3Ddevices, the stagnation line can act as a edge and facilitate cellrolling as explained below.

A stagnation line as defined herein is a region of zero flow velocitynear a surface of an object where flows on the surface converge fromdifferent directions. The shear along the stagnation line is zero, andthe flow velocity close to the surface defines a plane passing throughthe stagnation line. In this plane, the flow velocity must make an angleother than 90 degrees with respect to the stagnation line. The angle is90 degrees in the case of vertical posts).

In contemplated 3D devices of the invention, exterior surfaces arecoated with cell adhesion molecules that may induce cell rolling. Cellsin the fluid flowing across the surface may be induced to roll on thesurface. A cell rolling on the surface will roll towards the stagnationpoint, and then (under certain conditions) roll along the stagnationline and thereby follow it. Cells may roll in a direction at an angle tothe direction of fluid flow when the stagnation line is at an angle tothe direction of fluid flow. As in the case of rolling along a edge,cells may follow the stagnation line so long as the angle does notexceed a maximum angle α_(tr), whose value depends on the particularconditions of the cell separation system. The stagnation line may becurved depending on the surface under consideration and the flow fieldaround the surface.

OTHER EMBODIMENTS

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope of theinvention being indicated by the following claims.

We claim:
 1. A cell processing device comprising: a separation flowchamber, wherein the separation flow chamber comprises a surface that isat least partially coated with an ordered layer of cell adhesionmolecules, and the surface comprises at least one strip having an edgedefining a boundary between an area coated with the ordered layer andanother area that is not coated with the ordered layer; at least onecell inlet configured for flowing cells into the separation flowchamber; at least one buffer inlet for introducing a buffer stream thatdoes not contain cells into the separation chamber and configured toinduce cells entering separation chamber via the cell inlet to flow in aparallel direction of flow along with the buffer stream; wherein whencells having a surface moiety that is recognized by the cell adhesionmolecule are flowed through the separation chamber from the inlet to theoutlet, they roll along said edge at least part of the time in adirection that is at an angle α_(s) to the direction of flow; and atleast one outlet configured to accept at least one of the buffer streamand cells.
 2. The device of claim 1, wherein the surface that is atleast partially coated with an ordered layer of cell adhesion moleculesfurther comprises at least one strip that is at least partially coveredwith an ordered layer of cell adhesion molecules, said stripe disposedat an angle to the direction of flow between 5 and 45 degrees.
 3. Thedevice of claim 2, wherein the surface that is at least partially coatedwith an ordered layer of cell adhesion molecules further comprises aplurality of strips that are at least partially covered with an orderedlayer of cell adhesion molecules.
 4. The device of claim 3, wherein theplurality of strips each have at least one substantial linear edgeoriented at an angle relative to the flow direction.
 5. The device ofclaim 4, wherein the plurality of strips are separated from one anotherby a gap having a width, w_(g), in a range of from about 0.2 micrometersto about 10 millimeters.
 6. The device of claim 1, wherein the surfacethat is at least partially coated with an ordered layer of cell adhesionmolecules comprises a plurality of three dimensional surfaces that areat least partially covered with an ordered layer of cell adhesionmolecules.
 7. The device of claim 6, wherein the plurality of threedimensional surfaces are defined by shapes selected from squares,rectangles, triangles, polygons, ellipses, circles, arcs, waves, and anycombination thereof.
 8. The device of claim 1, further comprising atleast one additional outlet, at least one additional inlet, orcombinations thereof connected to the separation flow chamber.
 9. Thedevice of claim 1, wherein the separation flow chamber is defined bywalls having a height between about 5 μm and 1 mm.
 10. The device ofclaim 1, further comprising at least one collection channel forcollecting at least a subpopulation of cells that have been diverted bythe edge.
 11. The device of claim 10, further comprising markings alongthe at least one collection channel such that the height of a column ofcollected cells in the collection chamber gives an indication of thecell volume and/or of the cell count.
 12. The device of claim 1, furthercomprising a plurality of channels for collection of subpopulations ofcells flowed through the separation chamber.
 13. The device of claim 1,further comprising a pump for introducing cells into the device.
 14. Thedevice of claim 13, wherein the pump comprises a syringe pump.
 15. Thedevice of claim 1, wherein the cell adhesion molecules are selected fromthe group consisting of selectins, integrins, cadherins, immunoglobulincell adhesion molecules, and combinations thereof.
 16. The device ofclaim 1, wherein the surface that is at least partially coated with anordered layer of cell adhesion molecules defines at least one stagnationline and where flows on the surface can converge from differentdirections wherein when cells having a surface moiety that is recognizedby the cell adhesion molecule are flowed through the inlet to theoutlet, they roll at least part of the time along the stagnation line ina direction that is at an angle α_(s) to the direction of flow.
 17. Thedevice of claim 1, wherein the edge is characterized by a sharpnesscorresponding to a change from 10% to 90% density of cell adhesionmolecules over a distance of less than about 5 μm, wherein thepercentage density of cell adhesion molecules is measured as compared tothe maximum density of cell adhesion molecules in the coated areaadjacent to the edge.
 18. The device of claim 1, wherein the at leastone cell inlet defines a width that is less than a width of the at leastone buffer inlet.
 19. The device of claim 18 wherein the widths of thecell inlet and buffer inlet cooperate to provide a shear stress on cellsflowing over the surface that is in the range of 0.05 dyn/cm² to 50dyn/cm².
 20. The device of claim 18 wherein the widths of the cell inletand buffer inlet cooperate to provide a shear stress on cells flowingover the surface that is in the range of 0.2 dyn/cm² to 5 dyn/cm².